Pericycle-specific expression of microrna 167 in plants

ABSTRACT

Provided herein are compositions and methods for producing transgenic plants. In specific embodiments, transgenic plants comprise a construct comprising a polynucleotide encoding microRNA167 (miR167), or precursor thereof, operably linked to a plant pericycle-specific promote, wherein the miR167 is ectopically overexpressed in the transgenic plants, and wherein the promoter is optionally a constitutive or inducible promoter. In some embodiments, the transgenic plant has an improved agronomic or nutritional characteristic when cultivated in nitrogen-rich conditions as compared to a wild type plant cultivated in the same conditions. Also provided herein are commercial products (e.g., pulp, paper, paper products, or lumber) derived from the transgenic plants (e.g., transgenic trees) produced using the methods provided herein.

The present application claims priority benefits of U.S. ProvisionalApplication No. 60/918,443 filed Mar. 16, 2007, which is incorporated byreference herein in its entirety.

This invention was made in part with government support under Grantnumbers NIH NIGMS Grant GM3287; NSF Arabidopsis 2010 Genome GrantIBN0115586; and NSF Database Activities DBI-0445666. The government hascertain rights in the invention.

1. INTRODUCTION

Provided herein are compositions and methods for modulating nucleotidesequence expression, particularly for modulating gene expression inplants. In some embodiments, provided herein are compositions andmethods for genetically engineering plants to increase microRNAexpression in a specific tissue, such as in roots. In certain specificembodiments, a plant or tree is genetically engineered to alter (e.g.,increase) or constitutively express microRNA167 (also called “miR167”herein) in pericycle cells of the plant. In the presence of nitrogen,such genetically engineered plants can have one or more of the followingcharacteristics as compared to the wild-type counterpart: enhancedlateral root growth, enhanced surface area of roots, increased root massand/or increasing metabolic efficiency and nutrient uptake. Suchgenetically engineered plants can also, for example, grow larger, moreefficiently or rapidly, and/or have increased biomass. The geneticallyengineered plants can also have, in the presence of nitrogen, enhanceduptake of minerals or heavy metals in contaminated soils. The engineeredplants can be productively cultivated and increase lateral root growthunder conditions of nitrogen fertilizer input or in nitrogen rich soils.Alternatively, the engineered plants may be used to achieve fastergrowing or maturing crops or, higher crop yields and/or more nutritiousproducts even in nitrogen-rich cultivation conditions. In certainembodiments, the engineered plants and methods thereof are used in theproduction of commercial products. Some non-limiting example includegenetically engineered trees for e.g., the production of pulp, paper,paper products or lumber; tobacco, e.g., for the production ofcigarettes, cigars, or chewing tobacco; crops, e.g., for the productionof fruits, vegetables and other food, including grains, e.g., for theproduction of wheat, bread, flour, rice, corn; and soybean, canola,e.g., for the production of oils.

2. BACKGROUND

Plants exhibit remarkable developmental plasticity in response tochanging environments. This post-embryonic reorganization requirestranscriptional reprogramming at the cell-specific level to initiate neworgans to explore the soil for nutrients (see, e.g., Himanen et al.,2004, Proc Natl Acad Sci USA 101, 5146-51). Previous studies have showndistinct differences in the transcriptomes of Arabidopsis thaliana rootcells in steady state culture conditions (Birnbaum et al., 2003, Science302, 1956-60). However, little is known about the extent to which plantsmodulate gene expression at the cell specific level in response tochanging nutrient conditions.

Nitrate is a key required nutrient for the synthesis of amino acids,nucleotides and vitamins and is commonly considered to be the mostlimiting for normal plant growth (Vitousek et al., 1991, Biogeochemistry13:87-115). Nitrogenous fertilizer is usually supplied as ammoniumnitrate, potassium nitrate, or urea. Plants are keenly sensitive tonitrogen levels in the soil and, atypically of animal development, adopttheir body plan to cope with their environment (Lopez-Bucio et al.,2003, Curr Opin Plant Biol 6, 280-7); Malamy et al., 2005, Plant CellEnviron 28, 67-77); Walch-Liu et al., 2006, Ann Bot (Lond) 97, 875-81).For example, mutants in several general nitrogen (N)-assimilation genesaffect root architecture (Little et al., 2005, Proc Natl Acad Sci USA102, 13693-8; Remans et al., 2006, Proc Natl Acad Sci USA 103,19206-11). Transduction of this nitrogen signal is linked to a massiveand concerted gene expression response in the root (Gutierrez et al.,2007, Genome Biol 8, R7; Wang et al., 2003, Plant Physiol 132, 556-67).

Plant development is partially dependent on the plant's response to avariety of environmental signals. For example, the development of rootsystems is, in part, a response to the availability and distribution ofmoisture and nutrients within the soil.

In particular, lateral root development in Arabidopsis in response tonitrate is characterized by two distinct pathways. First, an increasedrate of lateral root elongation is a localized, direct response to thepresence of nitrate in the root zone. (Zhang et al., 1999, Proc NatlAcad Sci 96:6529-6534; Zhang and Forde, 2000, J of Exp. Bot. 51(342):51-59). In this aspect the nitrate ion appears to function as asignal rather than as a nutrient. (Zhang and Forde, 1998, Science279:407-409). Second, accumulation of high concentrations of nitrate andother nitrogen compounds in the shoot is correlated with a inhibition ofroot growth through a systemic effect on lateral root meristemactivation. (Zhang et al., 1999, supra).

Lateral root primordia formed on roots are the sites for lateral rootemergence. Nitrogen treatments of wild-type plants affects (e.g.,represses) the emergence of lateral roots. In wild-type plants, a largeproportion of root primordia emerge into lateral roots only innitrogen-poor conditions.

However, it would be advantageous to produce plants that would continueto increase lateral root growth, even in conditions of high nitrogencontent in the environment. By increasing lateral root growth oremergence, as well as, for example, enhanced surface area of roots,and/or increased root mass, such plants would be able to assimilate morenitrogen and uptake other essential growth nutrients from theenvironment (e.g., soil or water) that would otherwise be taken up atmuch lower rate. Thus, a need remains for plants whose lateral rootgrowth is insensitive to nitrogen content in its environment.

3. SUMMARY

A nitrogen-inducible gene in Arabidopsis, miR167, expressedpreferentially in roots, acts to specifically degrade the mRNA made fromother nitrogen-responsive regulatory genes responsible for therepression of lateral root development in the presence of nitrogen.Overexprssion of miR167 in the root meristem causes altered plantsensitivity to nitrate, and lateral root proliferation in nitrogen poorzones is increased. For example, miR167 overexpressing plants display anenhanced ratio of lateral root emergence in both nitrogen sufficient andnitrogen deplete conditions. These results indicate that miR167 is a keyregulator of developmental plasticity in Arabidopsis roots. Thus, themiR167 gene product is likely a component of the regulatory pathwaylinking external nitrogen availability to decreased lateral rootproliferation and Glu/Gln (the products of nitrate assimilation regulatelevels of miR167). While overexpression of miR167 in a plant increasesthe ratio of emerging:initating lateral roots, reduced overall levels oflateral roots are seen and plants are rendered sterile. Thus,overexpression of miR167 in only particular cells (e.g., pericyclecells) can overcome these effects while maintaining high levels oflateral root emergence. The overexpression of miRNA167 in pericyclecells also results in the downregulation of the genes listed in Table I,infra.

Manipulation of a nitrogen responsive molecule, such as miR167 inagronomic crops could be of value in maximizing plant utilization in thepresence of available nitrogen and in reducing agricultural nitrogeninputs, thereby providing economic and environmental benefits. Anotherbenefit would be the ability of the engineered plants to be productivelycultivated in both the presence and absence of nitrogen, such asfollowing nitrogen fertilization or in nitrogen-rich soil. Improvedcontrol of lateral root proliferation could have useful applications insoil remediation and in prevention of soil erosion. Increased rootbiomass may be beneficial in production of specific structuralcarbohydrates in the roots themselves, or in improving plant output ofspecialty compounds, including plastics, proteins, secondarymetabolites, and the like. Manipulation of nitrogen-responsive genes bymodulating miR167 levels could also be useful in stimulating rootproliferation of cuttings taken for plant propagation, especially inornamental and woody species. Additional improvements include morevigorous (i.e., faster) growth as well as greater vegetative and/orreproductive yield under normal cultivation conditions (i.e.,non-limiting nutrient conditions). To achieve these same improvements,traditional crop breeding methods would require screening largesegregating populations. The present invention circumvents the need forsuch large scale screening by producing plants many of which, if notmost, would have the desired characteristics.

We have discovered that miR167 levels are regulated by nitrogen nutrienttreatment and are a regulatory point for the control of lateral rootformation in plants, which is a key mechanism for plants to increasetheir surface area in the soil, to enhance nutrient acquisition. Based,in part, on this discovery, provided herein are compositions and methodsof manipulating miR167 expression in transgenic plants to optimizelateral root growth and/or nutrient acquisition in the soil without theneed for low nitrogen levels.

Compositions and methods are provided for modulating nucleotide sequenceexpression, particularly for modulating gene expression in plants. Thecompositions comprise precursor RNA constructs for the expression of anRNA precursor, such as miR167 precursor. In certain embodiments, aprecursor RNA construct comprises a promoter, such as a tissue specificpromoter, which is expressed in a plant cell, such as a pericycle cell,and promotes the expression of a precursor RNA having a miRNA, such asmiR167. The RNA precursor is cleaved in the plant cell to form anmiR167, which is a regulatory RNA that specifically controls geneexpression of certain target genes, which may, in turn, regulate avariety of other genes of the plant. The miR167 can be fully orpartially complementary to a portion of the nucleotide sequence encodinga target gene mRNA (e.g., ARF8) and functions to modulate expression ofthe target sequence or gene.

In certain embodiments, a precursor RNA construct is used in combinationwith a modulator to enhance the effect on gene expression. Modulatorsare proteins which can alter the level of at least one miRNA, such asmiR167, in a plant cell.

Any of a variety of promoters can be utilized in the constructs of theinvention depending on the desired outcome. Tissue-specific ortissue-preferred promoters, inducible promoters, developmentalpromoters, constitutive promoters and/or chimeric promoters can be usedto direct expression of the miRNA sequence or the modulator sequence inspecific cells or organs the plant, when fused to the appropriate cellor organ specific promoter.

Chimeric constructs expressing miR167 in transgenic plants (usingconstitutive or inducible promoters) can be used in the compositions andmethods provided herein to enhance lateral root formation, which in turnincrease nutrient uptake from soil. The use of inducible promoters can“prime” a plant to produce additional lateral roots for nutrientacquisition from the soil, for example, prior to fertilizer application.As minerals and nutrients rapidly leach out of soil, optimizing rootarchitecture to coincide with nutrient applications can enhance nutrientcapture from soil. This is especially true for negatively chargedminerals which bind poorly to negatively charged soil particles.

The discovery that miR167 regulates lateral root formation in responseto nitrogen treatment was only made possible by the use of cell-specifictranscript profiling as described in the examples herein. In certainembodiments, pericycle-specific promoters are used in the compositionsand methods provided herein to specifically express miR167.

In those embodiments, the overexpression of miR167 specifically in thepericycle can serve to increase the number of lateral roots, increasingthe surface area of roots, and make the root mass much more dense. Thisincreased root mass can enhance uptake of nitrogen and other nutrientsand water from the soil. The manipulation of miR167 levels in transgenicplants thus acts as a tool to increase metabolic efficiency in plantsand allows plants to better use smaller amounts of nitrogen and othermineral nutrients from the soil, reducing the quantities needed infertilizers, or show enhanced growth in the presence of normal or highlevels of nitrogen.

The present invention is based, in part, on the finding that miR167levels are regulated by nitrogen and are a regulatory point for thecontrol of lateral root formation in plants, and that increased orconstitutive miR167 expression in root-specific cells, such as thepericycle, results in enhanced lateral root growth, enhanced surfacearea of roots, increased root mass and/or increasing metabolicefficiency. The invention is illustrated herein by the way of a workingexample in which we used previously constructed Arabidopsis (model plantsystem) that had been engineered with recombinant constructs encoding astrong, constitutive plant promoter, the cauliflower mosaic virus (CaMV)35S promoter, operably linked with sequences encoding a miR167. RNA andprotein analyses showed that a majority of the engineered plantsexhibited ectopic, overexpression of miR167 (Wu et al., 2006,Development 133, 4211-8). The miR167 overexpressing transgenic lineshave a higher proportion of lateral root emergence and growth in thepresence of a nitrogen-rich environment than the control, wild-typeplant.

Alternatively, transgenic plants can include those plants that have beengenetically engineered to alter the expression of one or more or all ofthe genes listed in Table I, independent of miRNA167. In an alternativeembodiment, the present invention is directed to a transgenic plant inwhich one or more of the miRNA167-responsive genes listed in Table 1 isdown-regulated and which plant displays an enhanced ratio of lateralroot emergence in both nitrogen sufficient and nitrogen depletedconditions. Such a transgenic plant has one or more agronomic ornutritional characteristic including increased lateral root formation,increased surface area of roots, increased root mass, increasedmetabolic efficiency, increased nutrient uptake, faster growth rate,and/or greater fruit or seed yield (compared to the correspondingnon-transgenic plant. In a specific aspect, a transgenic plant of theinvention is one which has been genetically engineered such that one ormore, or all of the miRNA167-responsive genes listed in Table 1 havebeen downregulated. In one specific aspect of this embodiment, atransgenic plant of the invention is one which has been geneticallyengineered such that one or more, or all of the miRNA167-responsivetranscription factors and/or DNA binding proteins listed in Table 1 aredown-regulated, resulting in the desired agronomic and/or nutritionalcharacteristic.

4. TERMINOLOGY

Units, prefixes, and symbols may be denoted in their S1 accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxyl orientation, respectively. Numeric ranges recitedwithin the specification are inclusive of the numbers defining the rangeand include each integer within the defined range. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission. Nucleotides, likewise, may be referred to bytheir commonly accepted single-letter codes. Unless otherwise providedfor, software, electrical, and electronics terms as used herein are asdefined in The New IEEE Standard Dictionary of Electrical andElectronics Terms (5th edition, 1993). The terms defined below are morefully defined by reference to the specification as a whole.

As used herein, the term “agronomic” includes, but is not limited to,changes in root size, vegetative yield, seed yield or overall plantgrowth. Other agronomic properties include factors desirable toagricultural production and business.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,1993, American Society for Microbiology, Washington, D.C. The product ofamplification is termed an amplicon.

As used herein, “antisense orientation” includes reference to a duplexpolynucleotide sequence that is operably linked to a promoter in anorientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

In its broadest sense, a “delivery system,” as used herein, is anyvehicle capable of facilitating delivery of a nucleic acid (or nucleicacid complex) to a cell and/or uptake of the nucleic acid by the cell.

The term “ectopic” is used herein to mean abnormal subcellular (e.g.,switch between organellar and cytosolic localization), cell-type,tissue-type and/or developmental or temporal expression (e.g.,light/dark) patterns for the particular gene or enzyme in question. Suchectopic expression does not necessarily exclude expression in tissues ordevelopmental stages normal for said enzyme but rather entailsexpression in tissues or developmental stages not normal for the saidenzyme.

By “endogenous nucleic acid sequence” and similar terms, it is intendedthat the sequences are natively present in the recipient plant genomeand not substantially modified from its original form.

The term “exogenous nucleic acid sequence” as used herein refers to anucleic acid foreign to the recipient plant host or, native to the hostif the native nucleic acid is substantially modified from its originalform. For example, the term includes a nucleic acid originating in thehost species, where such sequence is operably linked to a promoter thatdiffers from the natural or wild-type promoter.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as are present in some plant, animal, andfungal mitochondria, the bacterium Mycoplasma capricolum, or the ciliateMacronucleus, may be used when the nucleic acid is expressed therein.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray et al., 1989, Nucl. Acids Res. 17:477-498). Thus, the maize preferred codon for a particular amino acidmay be derived from known gene sequences from maize. Maize codon usagefor 28 genes from maize plants is listed in Table 4 of Murray et al.,supra.

By “fragment” is intended a portion of the nucleotide sequence.Fragments of the modulator sequence will generally retain the biologicalactivity of the native suppressor protein. Alternatively, fragments ofthe targeting sequence may or may not retain biological activity. Suchtargeting sequences may be useful as hybridization probes, as antisenseconstructs, or as co-suppression sequences. Thus, fragments of anucleotide sequence may range from at least about 20 nucleotides, about50 nucleotides, about 100 nucleotides, and up to the full-lengthnucleotide sequence of the invention.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of, a native (non-synthetic), endogenous, biologically activeform of the specified protein. Methods to determine whether a sequenceis full-length are well known in the art including such exemplarytechniques as northern or western blots, primer extension, S1protection, and ribonuclease protection See, e.g., Plant MolecularBiology: A Laboratory Manual, Clark, Ed., 1997, Springer-Verlag, Berlin.Comparison to known full-length homologous (orthologous and/orparalogous) sequences can also be used to identify full-length sequencesof the present invention. Additionally, consensus sequences typicallypresent at the 5′ and 3′ untranslated regions of mRNA aid in theidentification of a polynucleotide as full-length. For example, theconsensus sequence ANNNNAUGG, where the underlined codon represents theN-terminal methionine, aids in determining whether the polynucleotidehas a complete 5′ end. Consensus sequences at the 3′ end, such aspolyadenylation sequences, aid in determining whether the polynucleotidehas a complete 3′ end.

The term “gene activity” refers to one or more steps involved in geneexpression, including transcription, translation, and the functioning ofthe protein encoded by the gene.

The term “genetic modification” as used herein refers to theintroduction of one or more exogenous nucleic acid sequences, e.g.,miR167 encoding sequences, as well as regulatory sequences, into one ormore plant cells, which can generate whole, sexually competent, viableplants. The term “genetically modified” or “genetically engineered” asused herein refers to a plant which has been generated through theaforementioned process. Genetically modified plants of the invention arecapable of self-pollinating or cross-pollinating with other plants ofthe same species so that the foreign gene, carried in the germ line, canbe inserted into or bred into agriculturally useful plant varieties.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,plant, insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with it as found in its naturalenvironment. The isolated material optionally comprises material notfound with the material in its natural environment; or (2) if thematerial is in its natural environment, the material has beensynthetically altered or synthetically produced by deliberate humanintervention and/or placed at a different location within the cell. Thesynthetic alteration or creation of the material can be performed on thematerial within or apart from its natural state. For example, anaturally-occurring nucleic acid becomes an isolated nucleic acid if itis altered or produced by non-natural, synthetic methods, or if it istranscribed from DNA which has been altered or produced by non-natural,synthetic methods. See, e.g., Compounds and Methods for Site DirectedMutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In vivoHomologous Sequence Targeting in Eukaryotic Cells; Zarling et al.,PCT/US93/03868. The isolated nucleic acid may also be produced by thesynthetic re-arrangement (“shuffling”) of a part or parts of one or moreallelic forms of the gene of interest. Likewise, a naturally-occurringnucleic acid (e.g., a promoter) becomes isolated if it is introduced toa different locus of the genome. Nucleic acids which are “isolated,” asdefined herein, are also referred to as “heterologous” nucleic acids.

As used herein, the term “marker” refers to a gene encoding a trait or aphenotype which permits the selection of, or the screening for, a plantor plant cell containing the marker.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer, or chimeras thereof, ineither single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism or of a tissuefrom that organism. Construction of exemplary nucleic acid libraries,such as genomic and cDNA libraries, is taught in standard molecularbiology references such as Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual, 2nd ed., Vol. 1-3; and Current Protocols in MolecularBiology, F. M. Ausubel et al., Eds., 1994, Current Protocols, a jointventure between Greene Publishing Associates, Inc. and John Wiley &Sons, Inc.

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

He term “orthologous” as used herein describes a relationship betweentwo or more polynucleotides or proteins. Two polynucleotides or proteinsare “orthologous” to one another if they serve a similar function indifferent organisms. In general, orthologous polynucleotides or proteinswill have similar catalytic functions (when they encode enzymes) or willserve similar structural functions (when they encode proteins or RNAthat form part of the ultrastructure of a cell).

The term “overexpression” is used herein to mean above the normalexpression level in the particular tissue, all and/or developmental ortemporal stage for said enzyme.

As used herein, the term “plant” is used in its broadest sense,including, but is not limited to, any species of woody, ornamental ordecorative, crop or cereal, fruit or vegetable plant, and algae (e.g.,Chlamydomonas reinhardtii). Non-limiting examples of plants includeplants from the genus Arabidopsis or the genus Oryza. Other examplesinclude plants from the genuses Acorus, Aegilops, Allium, Amborella,Antirrhinum, Apium, Arachis, Beta, Betula, Brassica, Capsicum,Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia,Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea,Lactuca, Linum, Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago,Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus,Physcomitrella, Picea, Pinus, Poncirus, Populus, Prunus, Robinia, Rosa,Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia,Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia.”Plants included in the invention are any plants amenable totransformation techniques, including gymnosperms and angiosperms, bothmonocotyledons and dicotyledons. Examples of monocotyledonousangiosperms include, but are not limited to, asparagus, field and sweetcorn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oatsand other cereal grains. Examples of dicotyledonous angiosperms include,but are not limited to tomato, tobacco, cotton, rapeseed, field beans,soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops orBrassica oleracea (e.g., cabbage, broccoli, cauliflower, brusselsprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash,melons, cantaloupe, sunflowers and various ornamentals. Examples ofwoody species include poplar, pine, sequoia, cedar, oak, etc. Stillother examples of plants include, but are not limited to, wheat,cauliflower, tomato, tobacco, corn, petunia, trees, etc. As used herein,the term “cereal crop” is used in its broadest sense. The term includes,but is not limited to, any species of grass, or grain plant (e.g.,barley, corn, oats, rice, wild rice, rye, wheat, millet, sorghum,triticale, etc.), non-grass plants (e.g., buckwheat flax, legumes orsoybeans, etc.). As used herein, the term “crop” or “crop plant” is usedin its broadest sense. The term includes, but is not limited to, anyspecies of plant or algae edible by humans or used as a feed for animalsor used, or consumed by humans, or any plant or algae used in industryor commerce. As used herein, the term “plant” also refers to either awhole plant, a plant part, or organs (e.g., leaves, stems, roots, etc.),a plant cell, or a group of plant cells, such as plant tissue, plantseeds and progeny of same. Plantlets are also included within themeaning of “plant.” The class of plants which can be used in the methodsof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants.

The term “plant cell” as used herein refers to protoplasts, gameteproducing cells, and cells which regenerate into whole plants. Plantcell, as used herein, further includes, without limitation, cellsobtained from or found in: seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores. Plant cells can alsobe understood to include modified cells, such as protoplasts, obtainedfrom the aforementioned tissues.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or chimeras or analogsthereof that have the essential nature of a natural deoxy- orribo-nucleotide in that they hybridize, under stringent hybridizationconditions, to substantially the same nucleotide sequence as naturallyoccurring nucleotides and/or allow translation into the same aminoacid(s) as the naturally occurring nucleotide(s). A polynucleotide canbe full-length or a subsequence of a native or heterologous structuralor regulatory gene. Unless otherwise indicated, the term includesreference to the specified sequence as well as the complementarysequence thereof. Thus, DNAs or RNAs with backbones modified forstability or for other reasons are “polynucleotides” as that term isintended herein. Moreover, DNAs or RNAs comprising unusual bases, suchas inosine, or modified bases, such as tritylated bases, to name justtwo examples, are polynucleotides as the term is used herein. It will beappreciated that a great variety of modifications have been made to DNAand RNA that serve many useful purposes known to those of skill in theart. The term polynucleotide as it is employed herein embraces suchchemically-, enzymatically- or metabolically-modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a correspondingnaturally-occurring amino acid, as well as to naturally-occurring aminoacid polymers. The essential nature of such analogues ofnaturally-occurring amino acids is that, when incorporated into aprotein, that protein is specifically reactive to antibodies elicited tothe same protein but consisting entirely of naturally occurring aminoacids. The terms “polypeptide”, “peptide” and “protein” are alsoinclusive of modifications including, but not limited to, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation. Further, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria which comprise genes expressed inplant cells such Agrobacterium or Rhizobium. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, or seeds. Suchpromoters are referred to as “tissue preferred.” Promoters whichinitiate transcription only in certain tissue are referred to as “tissuespecific.” A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “repressible” promoter is apromoter which is under environmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue specific, tissuepreferred, cell type specific, and inducible promoters represent theclass of “non-constitutive” promoters. A “constitutive” promoter is apromoter which is active under most environmental conditions.

As used herein “recombinant” includes reference to a cell or vector thathas been modified by the introduction of a heterologous nucleic acid, orto a cell derived from a cell so modified. Thus, for example,recombinant cells express genes that are not found in identical formwithin the native (non-recombinant) form of the cell, or exhibit alteredexpression of native genes, as a result of deliberate humanintervention. The term “recombinant” as used herein does not encompassthe alteration of the cell or vector by events (e.g., spontaneousmutation, natural transformation, transduction, or transposition)occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The term “regulatory sequence” as used herein refers to a nucleic acidsequence capable of controlling the transcription of an operablyassociated gene. Therefore, placing a gene under the regulatory controlof a promoter or a regulatory element means positioning the gene suchthat the expression of the gene is controlled by the regulatorysequence(s). Because a microRNA binds to its target, it is a posttranscriptional mechanism for regulating levels of mRNA. Thus, an miRNA,e.g., miR167, can also be considered a “regulatory sequence” herein. Notjust transcription factors.

The term “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally occurring amino acids.

The term “root-specific promotor” is a polynucleotide encoding apromoter that specifically binds to transcription factors primarily oronly in roots.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, preferably 90% sequenceidentity, and most preferably 100% sequence identity (i.e.,complementary) with each other.

As used herein, a “stem-loop motif” or a “stem-loop structure,”sometimes also referred to as a “hairpin structure,” is given itsordinary meaning in the art, i.e., in reference to a single nucleic acidmolecule having a secondary structure that includes a double-strandedregion (a “stem” portion) composed of two regions of nucleotides (of thesame molecule) forming either side of the double-stranded portion, andat least one “loop” region, comprising uncomplemented nucleotides (i.e.,a single-stranded region).

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence, to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, 1984, Anal. Biochem., 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen, 1993,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York; and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., 1995, GreenePublishing and Wiley-Interscience, New York. Hybridization and/or washconditions can be applied for at least 10, 30, 60, 90, 120, or 240minutes.

As used herein, “transcription factor” includes reference to a proteinwhich interacts with a DNA regulatory element to affect expression of astructural gene or expression of a second regulatory gene.“Transcription factor” may also refer to the DNA encoding saidtranscription factor protein. The function of a transcription factor mayinclude activation or repression of transcription initiation.

The term “transfection,” as used herein, refers to the introduction of anucleic acid into a cell, for example, a precursor miRNA, or anucleotide sequence able to be transcribed to produce precursor miRNA.

As used herein, the term “transformation” means alteration of thegenotype of a host plant by the introduction of miR167-nucleic acidsequence.

As used herein, “transgenic plant” includes reference to a plant whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used inintroduction of a polynucleotide of the present invention into a hostcell. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween a polynucleotide/polypeptide of the present invention with areference polynucleotide/polypeptide: (a) “reference sequence”, (b)“comparison window”, (c) “sequence identity”, and (d) “percentage ofsequence identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison with a polynucleotide/polypeptide of thepresent invention. A reference sequence may be a subset or the entiretyof a specified sequence; for example, as a segment of a full-length cDNAor gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” includes reference to acontiguous and specified segment of a polynucleotide/polypeptidesequence, wherein the polynucleotide/polypeptide sequence may becompared to a reference sequence and wherein the portion of thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acids residues in length, andoptionally can be 30, 40, 50, 100, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide/polypeptide sequence, a gappenalty is typically introduced and is subtracted from the number ofmatches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, 1981, Adv. Appl.Math. 2: 482; by the homology alignment algorithm of Needleman andWunsch, 1970, J. Mol. Biol. 48: 443; by the search for similarity methodof Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. 85: 2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, 1988, Gene 73: 237-244; Higgins and Sharp, 1989,CABIOS 5: 151-153; Corpet et al., 1988, Nucleic Acids Research 16:10881-90; Huang et al., 1992, Computer Applications in the Biosciences8: 155-65; and Pearson et al., 1994, Methods in Molecular Biology 24:307-331.

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel et al., Eds., 1995, GreenePublishing and Wiley-Interscience, New York.

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information (world-wideweb at ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold. These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, 1993, Proc. Natl. Acad.Sci. USA 90:5873-5877). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequenceswhich may be homopolymeric tracts, short-period repeats, or regionsenriched in one or more amino acids. Such low-complexity regions may bealigned between unrelated proteins even though other regions of theprotein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen, 1993, Comput. Chem., 17:149-163)and XNU (Claverie and States, 1993, Comput. Chem., 17:191-201)low-complexity filters can be employed alone or in combination.

Unless otherwise stated, nucleotide and protein identity/similarityvalues provided herein are calculated using GAP (GCG Version 10) underdefault values.

GAP (Global Alignment Program) can also be used to compare apolynucleotide or polypeptide of the present invention with a referencesequence. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.48: 443-453, 1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 100. Thus, for example, the gapcreation and gap extension penalties can each independently be: 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff & Henikoff, 1989, Proc. Natl. Acad.Sci. USA 89:10915).

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp, 1989, CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, 1988, Computer Applic. Biol. Sci.,4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

Polynucleotide sequences having “substantial identity” are thosesequences having at least about 50%, 60% sequence identity, generally70% sequence identity, preferably at least 80%, more preferably at least90%, and most preferably at least 95%, compared to a reference sequenceusing one of the alignment programs described above. Preferably sequenceidentity is determined using the default parameters determined by theprogram. Substantial identity of amino acid sequences generally meanssequence identity of at least 50%, more preferably at least 70%, 80%,90%, and most preferably at least 95%. Nucleotide sequences aregenerally substantially identical if the two molecules hybridize to eachother under stringent conditions.

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

As used herein, the term “transgenic,” when used in reference to a plant(i.e., a “transgenic plant”) refers to a plant that contains at leastone heterologous gene in one or more of its cells.

As used herein, “substantially complementary,” in reference to nucleicacids, refers to sequences of nucleotides (which may be on the samenucleic acid molecule or on different molecules) that are sufficientlycomplementary to be able to interact with each other in a predictablefashion, for example, producing a generally predictable secondarystructure, such as a stem-loop motif. In some cases, two sequences ofnucleotides that are substantially complementary may be at least about75% complementary to each other, and in some cases, are at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, at least about 99.5%, or 100% complementary to each other. In somecases, two molecules that are sufficiently complementary may have amaximum of 40 mismatches (e.g., where one base of the nucleic acidsequence does not have a complementary partner on the other nucleic acidsequence, for example, due to additions, deletions, substitutions,bulges, etc.), and in other cases, the two molecules may have a maximumof 30 mismatches, 20 mismatches, 10 mismatches, or 7 mismatches. Instill other cases, the two sufficiently complementary nucleic acidsequences may have a maximum of 0, 1, 2, 3, 4, 5, or 6 mismatches.

By “variants” is intended substantially similar sequences. For “variant”nucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of the modulator of the invention. Variant nucleotidesequences include synthetically derived sequences, such as thosegenerated, for example, using site-directed mutagenesis. Generally,variants of a particular nucleotide sequence of the invention will haveat least about 40%, 50%, 60%, 65%, 70%, generally at least about 75%,80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, and more preferably at least about 98%, 99% or more sequenceidentity to that particular nucleotide sequence as determined bysequence alignment programs described elsewhere herein using defaultparameters. By “variant” protein is intended a protein derived from thenative protein by deletion or addition of one or more amino acids to theN-terminal and/or C-terminal end of the native protein; deletion oraddition of one or more amino acids at one or more sites in the nativeprotein; or substitution of one or more amino acids at one or more sitesin the native protein. Such variants may result from, for example,genetic polymorphism or human manipulation. Conservative amino acidsubstitutions will generally result in variants that retain biologicalfunction

As used herein, the term “yield” or “plant yield” refers to increasedplant growth, and/or increased biomass. In one embodiment, increasedyield results from increased growth rate and increased root size. Inanother embodiment, increased yield is derived from shoot growth. Instill another embodiment, increased yield is derived from fruit growth.

5. DESCRIPTION OF THE FIGURES

FIGS. 1A-E shows that the N-response is highly cell specific. (A)N-response clusters. The number of genes within each cluster isindicated using the length of the adjacent bar. Clusters that exhibitsome degree of Glu/Gln-responsiveness are indicated with hatching on thebars. These were determined by their partial loss of N-responsiveness onaddition of MSX and the regaining of this on Glu/Gln resupply. (B)Average log 2 microarray expression values for the three largest generesponse clusters in control and treated experiments. Top panel: genecluster that is N-induced in all cell populations contains anover-representation of GO terms relating to control of metabolism.Middle panel: gene cluster that is N-induced in the lateral root cap andpericycle contains an over-representation of the GO term ‘regulation oftranscription’. Bottom panel: gene cluster that is N-repressed in theepidermis and stele contains an over-representation of the GO term‘photosynthesis.’ (C) Schematic of the Arabidopsis root showing the fivecell populations studied: red, LRC; dark blue/light blue,epidermis/cortex; orange, endodermis; green, pericycle; green/yellow,stele (pericycle and vascular tissues). The direction of nitrate uptakeand its assimilation into amino acids (aa) is indicated using arrows.

FIGS. 2A-B shows miR167 and ARF8 are N-regulated in an opposite anddependent fashion to modulate lateral root development. (A) Network ofgenes that are induced in the pericycle cell layer. (B) Zoom-in of thispericycle N-induced network to show a sub-network controlled by ARF8 andmiR167. ARF6 (At1g30330) was not found to be significantly N-regulatedin this study but is included in the network since its is a knownpartner of ARF8 (Wu et al., 2006, Development 133, 4211-8); ARF6 doesappear to be N-regulated in a similar fashion to ARF8 when the rawmicroarray data is viewed thus it's inclusion is valid. ARF8 targetgenes include: Transcription factors (diamonds) or DNA-binding proteins(squares with circle pattern): (1a) At3g61310, (1b) At1g76420, (1c)At1g24260, (1d) At1g79350, (1e) At1g63470, (1f) At2g20100, (1g)At3g45610. LRR kinases (squares with circle pattern): (2a) At2g26330,(2b) At3g57830, (2c) At2g01210. Metabolic genes (black squares): (3a)At3g16170, (3b) At1g48100, (3c) At1g11730, (3d) At1g70710 (CEL1), (3e)At1g32930. Expressed proteins (squares with crisscross pattern): (4a)At3g13000, (4b) At2g38160, (4c) At1g03170, (4d) At3g13510, (4e)At2g23700, (4f) At3g11000. Other cellular function: (5) Kinesin motorprotein-related At3g10310. (6) DNA polymerase delta smallsubunit-related At2g42120. (7) Cyclin A2; 3 At1g15570. (8)Calmodulin-binding family protein At2g26180. (9) DNA primase At1g67320.(10) Emsy N terminus domain-containing protein/ENT domain-containingprotein At2g44440. In addition, At3g61310 and ARF6 (At1g30330) are alsopredicted miR167 targets (according to Dezulian et al., 2006,Bioinformatics 22, 359-60). White box denotes genes that are notspecifically Glu-responsive.

FIGS. 3A-N show antagonistic regulation between miRNA167 and ARF-8 inresponse to nitrogen mediates lateral root initiation and emergence. A,D, G, GUS-stained control roots. B, E, H, GUS-stained nitrate-treatedroots; all roots were GUS-stained for 12 hours. C, F, I, Averageexpression level of indicated genes and constructs assessed by qPCR inwhole roots control-treated (C) or nitrate-treated (T) from 3 biologicalreplicates. A-C, Nitrogen-induction of gARF8:GUS and qPCR quantificationof ARF8 expression. D-F, Nitrogen-repression of PMIR167a::GUS and qPCRquantification of pre-miR167a expression. A control microRNA (miR160)showed no nitrogen response. G-I, Loss of nitrogen induction of ARF8expression in mARF8:GUS and qPCR quantification of GUS expression. J,Response of mature miR167a/b to nitrogen treatment in the five cellpopulations profiled. K, Confocal images of initiating and pre-emergentlateral roots (GFP-marked) and emerging lateral roots (not GFP-marked)in the line used for cell sorting that marks pericycle cells adjacent tothe xylem pole (E3754). L, Bar graphs show the relative mean percentagesof initiating (light colored bars) and emerging (dark colored bars)lateral roots in Co1-0, arf8, and P35S::MIR167a four days after 12day-old seedlings were either mock-treated (no treatment) ornitrate-treated. To the right of the bars, the average number of lateralroots per seedling is shown. The P35S::MIR167a has fewer lateral rootsin total as a consequence of having shorter roots. Co10 (n=21), arf8-3(n=11), and 35S::miR167a (n=6) 4 days after 12 day-old seedlings wereeither control-treated (no treatment) or N-treated. Co10 (top two bars)control-treatment 32%±3 initiating, 68%±3 emerging; N-treatment 48±3initiating, 52±3 emerging. 35S::miR167a (middle two bars)control-treatment 23%±3 initiating, 77%±7 emerging; N-treatment 19%±4initiating, 810%±9 emerging. arf8 (bottom two bars) control-treatment23%±3 initiating, 77%±4 emerging; N-treatment 34±3 initiating, 66±4emerging. On the right of the bars the average number of lateral rootsper seedling is shown. 35S::miR167-expressing seedlings have fewerlateral roots in total at the same time as having a higher percentage ofemerging lateral roots. 35S::miR167-expressing seedlings are insensitiveto nitrogen-downregulation of lateral root emergence.35S::miR167-expressing seedlings also exhibit fewer lateral rootsoverall. arf8 seedlings have a reduced N-sensitivity. We used achi-squared test to compare the wild-type ratio of initiating:emerginglateral roots with the ratios that we observed in 35S::miR167 and arf8.M, Heat map showing the response (blue=induction, yellow=repression) ofARF8 and the 126 predicted target genes in the putative ARF8 module toKNO3, KCl, MSX and Gln treatments in sorted pericycle founder cells. N,Summary of the miR167/ARF8-regulated genetic circuitry that controls thebalance between initiating and emerging lateral roots in relation tonitrogen availability. Scale bars: 25 μm.

FIG. 4 depicts ARF8 cluster genes

FIG. 5 depicts signal values for the 25 cell-specific genes

FIG. 6A-C shows that cell sorting enhances sensitivity to discoverN-regulation of genes. qPCR confirms the N-response found in individualcell populations using microarrays that was not found at the whole rootlevel. (A) At5g03280 is induced in the LRC cell population. (B)At5g22300 is depressed in the epidermis/cortex cell population. (C)At3g61310 is induced in the pericycle cell population. n=3 biologicalreplicates.

FIGS. 7A-D depicts the expression patterns of four of the five GFPmarker lines used in this study. (A) E4722 marks the lateral root cap;(B) E1001 marks the epidermis and cortex; (C) E470 is expressed in theendodermis and pericycle; (D) E3754 marks the pericycle. The fifth lineused, pWOL::GFP, is expressed specifically in the stele from thepromeristem to the early differentiation stages (published in ²⁰). Scalebars: 25 μm.

6. DETAILED DESCRIPTION

A nitrogen-inducible gene in Arabidopsis, miR167, expressedpreferentially in roots, acts to specifically degrade the mRNA made fromother nitrogen-responsive regulatory genes responsible for therepression of lateral root development in the presence of nitrogen.Overexprssion of miR167 in the root meristem causes altered plantsensitivity to nitrate, and lateral root proliferation in nitrogen poorzones is increased. For example, miR167 overexpressing plants display anenhanced ratio of lateral root emergence in both nitrogen sufficient andnitrogen deplete conditions. These results indicate that miR167 is a keyregulator of developmental plasticity in Arabidopsis roots. Thus, themiR167 gene product is likely a component of the regulatory pathwaylinking external nitrogen availability to decreased lateral rootproliferation and Glu/Gln (the products of nitrate assimilation regulatelevels of miR167). While overexpression of miR167 in a plant increasesthe ratio of emerging:initating lateral roots, reduced overall levels oflateral roots are seen and plants are rendered sterile. Thus,overexpression of miR167 in only particular cells (e.g., pericyclecells) can overcome these effects while maintaining high levels oflateral root emergence.

Manipulation of a nitrogen responsive molecule, such as miR167 inagronomic crops could be of value in maximizing plant utilization in thepresence of available nitrogen and in reducing agricultural nitrogeninputs, thereby providing economic and environmental benefits. Anotherbenefit would be the ability of the engineered plants to be productivelycultivated in both the presence and absence of nitrogen, such asfollowing nitrogen fertilization or in nitrogen-rich soil. Improvedcontrol of lateral root proliferation could have useful applications insoil remediation and in prevention of soil erosion. Increased rootbiomass may be beneficial in production of specific structuralcarbohydrates in the roots themselves, or in improving plant output ofspecialty compounds, including plastics, proteins, secondarymetabolites, and the like. Manipulation of nitrogen-responsive genes bymodulating miR167 levels could also be useful in stimulating rootproliferation of cuttings taken for plant propagation, especially inornamental and woody species. Additional improvements include morevigorous (i.e., faster) growth as well as greater vegetative and/orreproductive yield under normal cultivation conditions (i.e.,non-limiting nutrient conditions). To achieve these same improvements,traditional crop breeding methods would require screening largesegregating populations. The present invention circumvents the need forsuch large scale screening by producing plants many of which, if notmost, would have the desired characteristics.

We have discovered that miR167 levels are regulated by nitrogen nutrienttreatment and are a regulatory point for the control of lateral rootformation in plants, which is a key mechanism for plants to increasetheir surface area in the soil, to enhance nutrient acquisition. Based,in part, on this discovery, provided herein are compositions and methodsof manipulating miR167 expression in transgenic plants to optimizelateral root growth and/or nutrient acquisition in the soil without theneed for low nitrogen levels.

Compositions and methods are provided for modulating nucleotide sequenceexpression, particularly for modulating gene expression in plants. Thecompositions comprise precursor RNA constructs for the expression of anRNA precursor, such as miR167 precursor. In certain embodiments, aprecursor RNA construct comprises a promoter, such as a tissue specificpromoter, which is expressed in a plant cell, such as a pericycle cell,and promotes the expression of a precursor RNA having a miRNA, such asmiR167. The RNA precursor is cleaved in the plant cell to form anmiR167, which is a regulatory RNA that specifically controls geneexpression of certain target genes, which may, in turn, regulate avariety of other genes of the plant. The miR167 can be fully orpartially complementary to a portion of the nucleotide sequence encodinga target gene mRNA (e.g., ARF8) and functions to modulate expression ofthe target sequence or gene. Thus, an RNA precursor construct can bedesigned to modulate levels of any mRNA nucleotide sequence of interest,either an endogenous plant mRNA or alternatively a transgene mRNA. TheRNA precursor can also be designed to produce a transcript that isprocessed via the miRNA pathway to produce an miRNA complementary to aportion of mRNA, the target mRNA, that corresponds to the target gene.The miRNA modulates the expression of the target gene, such as byaltering the production, processing, stability, or translation of thetarget mRNA and thereby altering the expression of the target mRNAproduct.

In certain embodiments, a precursor RNA construct is used in combinationwith a modulator to enhance the effect on gene expression. Modulatorsare proteins which can alter the level of at least one miRNA, such asmiR167, in a plant cell, including, but not limited to plant and viralproteins that are known to alter RNA silencing. Expression of amodulator in the presence of the precursor RNA alters the accumulationof miRNAs and thus enhances the regulatory capabilities of miRNAs. Inthis manner, a plant expressing both the precursor RNA and a modulatorcan be constructed to modulate expression of a target gene.

Any of a variety of promoters can be utilized in the constructs of theinvention depending on the desired outcome. Tissue-specific ortissue-preferred promoters, inducible promoters, developmentalpromoters, constitutive promoters and/or chimeric promoters can be usedto direct expression of the miRNA sequence or the modulator sequence inspecific cells or organs the plant, when fused to the appropriate cellor organ specific promoter.

Chimeric constructs expressing miR167 in transgenic plants (usingconstitutive or inducible promoters) can be used in the compositions andmethods provided herein to enhance lateral root formation, which in turnincrease nutrient uptake from soil. The use of inducible promoters can“prime” a plant to produce additional lateral roots for nutrientacquisition from the soil, for example, prior to fertilizer application.As minerals and nutrients rapidly leach out of soil, optimizing rootarchitecture to coincide with nutrient applications can enhance nutrientcapture from soil. This is especially true for negatively chargedminerals which bind poorly to negatively charged soil particles.

The discovery that miR167 regulates lateral root formation in responseto nitrogen treatment was only made possible by the use of cell-specifictranscript profiling as described in the examples herein. In certainembodiments, pericycle-specific promoters are used in the compositionsand methods provided herein to specifically express miR167.

In those embodiments, the overexpression of miR167 specifically in thepericycle can serve to increase the number of lateral roots, increasingthe surface area of roots, and make the root mass much more dense. Thisincreased root mass can enhance uptake of nitrogen and other nutrientsand water from the soil. The manipulation of miR167 levels in transgenicplants thus acts as a tool to increase metabolic efficiency in plantsand allows plants to better use smaller amounts of nitrogen and othermineral nutrients from the soil, reducing the quantities needed infertilizers, or show enhanced growth in the presence of normal or highlevels of nitrogen.

Achieving the desired plant improvements may require, in some instances,the ectopic overexpression of a miR167 in specific organs or cell types,such as in pericycle cells of a plant. The modified expression mayinvolve engineering the plant with any or several of the following: a) atransgene in which the coding sequence for the miRNA is operablyassociated to a strong, constitutive promoter; b) additional copies ofthe native gene encoding the desired miR167; c) regulatory gene(s) thatactivates the expression of miR167; d) a copy of the native miR167 genethat has its regulatory region modified for enhanced expression; and e)a transgene which expresses a mutated, altered or chimeric version of amiR167. In certain embodiments, the miR167 gene or transgene is underthe control of a constitutive or inducible promoter, and in specificembodiments the promoter is a pericycle-specific promoter.

In other instances, achieving the desired plant improvements may requirealtering the expression pattern of a miR167. The altered expressionpattern may involve engineering the plant with any or many of thefollowing: a) a transgene in which the coding sequence for the miR167 isoperably associated to a promoter with the desired expression pattern(such promoters may include those considered to have tissue (e.g.,pericycle) or developmental-specific expression patterns); b) modifiedregulatory genes that activates the expression of the miR167-encodinggene in the preferred pattern; c) a native copy of the miR167-encodinggene that has its regulatory region modified to express in the preferredpattern. In certain embodiments, the miR167 gene is under the control ofa constitutive or inducible promoter, and in specific embodiments thepromoter is a pericycle-specific promoter.

In still other instances, achieving the desired plant improvements mayrequire expressing altered or different forms of miR167. Such effortsmay involve developing a plant-expressible gene encoding a miRNA167 withproperties different from those of the corresponding host plant miR167and engineering plants with that gene construct. Gene sequences encodingsuch miR167 may be obtained from a variety of sources, including, butnot limited to bacteria, yeast, algae, animals, and plants. In somecases, such coding sequences may be directly used in the construction ofplant-expressible gene fusions by operably linking the sequence with adesired plant-active promoter. In other cases, the utilization of suchcoding sequences in gene fusions may require prior modification by invitro mutagenesis or de novo synthesis to enhance their translatabilityin the host plant or to alter the properties of the miR167 encodedthereon. Useful alterations may include, but are not limited to,modifications of residues involved in target mRNA binding.

A plant with the desired improvement can be isolated by screening theengineered plants for altered expression pattern or level of the miR167(or precursor thereof) and/or expression pattern or level of a direct orindirect target polynucleotide of the miR167, such as mRNA for ARF8, ordownstream gene products whose expression is modulated by ARF8 (FIG.2B), such as At3g61310, At1g76420, At1g24260, At1g79350, At1g63470,At2g20100, At3g45610; At2g26330, At3g57830, At2g01210; At3g16170,At1g48100, At1g11730, At1g70710 (CEL1), At1g32930, At3g13000, At2g38160,At1g03170, At3g13510, At2g23700, At3g11000, At3g10310, At2g42120,At1g15570, At2g26180, At1g67320 and/or At2g44440. A plant can also bescreened for lateral root growth, root surface area, root biomass,nutrient uptake, overall increased plant growth rate, enhancedvegetative yield, or improved reproductive yields. The screening of theengineered plants can involve Southern analysis to confirm the presenceand number of transgene insertions, Northern analysis, RNase protection,primer extension, reverse transcriptase/PCR and the like to measure mRNAlevels; measuring the amino acid composition, free amino acid pool ortotal nitrogen content of various plant tissues; monitoring numbers andtypes of lateral root primordia and lateral roots; measuring growthrates in terms of fresh weight gains over time; or measuring plant yieldin terms of total dry weight and/or total seed weight, or a combinationof any of the above methods.

The present invention is based, in part, on the finding that miR167levels are regulated by nitrogen and are a regulatory point for thecontrol of lateral root formation in plants, and that increased orconstitutive miR167 expression in root-specific cells, such as thepericycle, results in enhanced lateral root growth, enhanced surfacearea of roots, increased root mass and/or increasing metabolicefficiency. The invention is illustrated herein by the way of a workingexample in which we used previously constructed Arabidopsis (model plantsystem) that had been engineered with recombinant constructs encoding astrong, constitutive plant promoter, the cauliflower mosaic virus (CaMV)35S promoter, operably linked with sequences encoding a miR167. RNA andprotein analyses showed that a majority of the engineered plantsexhibited ectopic, overexpression of miR167 (Wu et al., 2006,Development 133, 4211-8). The miR167 overexpressing transgenic lineshave a higher proportion of lateral root emergence and growth in thepresence of a nitrogen-rich environment than the control, wild-typeplant.

The present invention provides methods for increasing the yield of aplant, such as a agricultural crop, such as by increasing (e.g., byoverexpressing and/or inducibly or constitutively expressing) miR167expression levels in the root of a plant. Increasing miRNA expressionlevel in plant root cells results in increased lateral root growth, rootbiomass, nutrient uptake, overall plant growth and yield, even in thepresence of nitrogen.

In a preferred embodiment, the invention provides a method for producinga genetically modified plant characterized as having increased lateralroot growth, root biomass, nutrient uptake, overall plant growth and/oryield as compared to a plant which has not been genetically modified(e.g., a wild-type plant), particularly when the transgenic plant isgrown in the presence of nitrogen. In specific embodiments, the methodcomprises contacting plant cells with nucleic acid encoding a miR167,wherein the nucleic acid is operably associated with a regulatorysequence, such as a tissue-specific (e.g., a pericycle-specific)promoter, to obtain transformed plant cells; producing plants from thetransformed plant cells; and thereafter selecting a plant exhibitingincreased lateral root growth, root biomass, nutrient uptake, overallplant growth and/or yield as compared to a plant which has not beengenetically modified (e.g., a wild-type plant), particularly when thetransgenic plant is grown in a nitrogen-moderate or nitrogen-richenvironment (e.g., soil treated with a fertilizer).

In some embodiments, a regulatory sequence, such as a promoter, usefulin the compositions and methods provided herein can be derived from anyknown pericycle-specific gene or the orthologous gene from any otherplant species using methods currently known in the art or describedelsewhere herein. However, functional fragments of the selectedregulatory sequence may also be used which confer a modifiedtranscriptional activity upon nucleic acid sequence which are operablylinked to the regulatory sequence. By “modified transcriptionalactivity” is meant transcription of linked sequences above or belowwild-type expression of the linked sequence.

6.1 miRNAs

miRNAs are a large class of about 21- to 24-nucleotide noncoding,regulatory RNAs, which are found not only in plants, but also innematodes, Drosophila, and humans. There are many miRNA genes, whichhave different patterns of expression patterns dependant on thetissue-type and stage of development. When these miRNAs are expressed,they pair to sites within the 3′ untranslated region (“UTR”) of targetmRNAs, triggering the translational repression of the mRNA targets. Bycontrast to animals, the miRNA target sites in plants are generallywithin the coding sequence. miRNAs are single-stranded, and theiraccumulation is developmentally regulated. They derive from partiallydouble-stranded precursor RNAs that are transcribed from genes that donot encode protein. Most of the miRNAs (in animals) lack completecomplementarity to any putative target mRNA, but were thought to perhapsregulate gene expression during development, perhaps at the level ofdevelopment. In plants, complete complementarity of miRNAs to theirtarget is more common.

As used herein, a “microRNA” or an “miRNA” is given its ordinary meaningin the art. Typically, an miRNA is a RNA molecule derived from genomicloci processed from transcripts that can form local RNA precursor miRNAstructures. The mature miRNA usually has 20 to 24 nucleotides, althoughin some cases, other numbers of nucleotides may be present (for example,between 18 and 26 nucleotides). miRNAs are usually detectable onNorthern blots. The miRNA has the potential to pair to flanking genomicsequences, placing the mature miRNA within an imperfect RNA duplex whichmay be needed for its processing from a longer precursor transcript. Inaddition, miRNAs are typically derived from a segment of the genome thatis distinct from predicted protein-coding regions. As used herein,“plant-derived” miRNA is miRNA that is produced using precursor miRNAsexpressed naturally in a plant cell. For instance, the miRNA precursor,or at least a portion thereof (for example, a hairpin or stem-loopmotif, as further discussed below), can be expressed from a native plantgene.

miRNA is typically produced through the processing of precursor miRNA.Thus, in certain embodiments, a precursor miRNA is processed to producemiRNA in a plant cell. In specific embodiments, the precursor miRNA is aprecursor MiR167 that is processed to produce miR167 in a plant cell(such as a pericycle cell). Additionally, the precursor miRNA may beisolated, e.g., from plant cells, according to certain embodiments. Asused herein, “precursor miRNA” is generally composed of any type ofnucleic acid-based molecules capable of accommodating miRNA sequencesand stem-loop motifs incorporating the miRNA sequences. The precursormiRNA, such as a precursor miR167, may be naturally or artificiallygenerated. Typically, the precursor miRNA molecule is an isolatednucleic acid having a stem-loop structure and a miRNA sequenceincorporated therein. The miRNA sequences and the sequences includingthe stem-loop motifs do not all necessarily have to originate from thesame organism. In some embodiments, the primary sequence of theprecursor miRNA, exclusive of the miRNA, is derived from naturalsequences flanking plant-derived miRNAs, such as miR167.

The compositions provided herein comprise precursor RNA constructs forthe expression, and preferably the overexpression and/or inducibleexpression or overexpression, of an RNA precursor, such as miR167precursor. The precursor RNA construct can comprise a promoter that isexpressed in a plant cell driving the expression of a nucleotidesequence that encodes the precursor RNA having a miRNA. The RNAprecursor can be cleaved in a plant cell to form the miRNA. The miRNA iscomplementary to a portion of a target gene or nucleotide sequence andfunction to modulate expression of the target sequence or gene, (e.g.,ARF8) and/or indirectly modulate expression or repression of downstreamgenes that are regulated by the target gene. The precursor RNAconstructs are designed to direct the expression in the plant an RNAprecursor that has an miRNA that is complementary to a portion of atarget nucleotide sequence. Such precursor RNAs, their respective miRNAsand the genes that encode them are known in the art and have beenidentified in plants. See, e.g., Reinhart et al., 2002, Genes &Development 16:1616-1626, Llave et al., 2002, Plant Cell 14:1605-1619,and Wu et al., 2006, Development, 133.4211. The nucleotide sequence thatencodes the precursor RNA can comprise an miRNA region that iscomplementary to a portion of the target gene. The regions which flankthe miRNA region are selected from the sequences known in the art formiRNA precursors, particularly plant miRNA precursors, more particularlythose plant miRNA precursors disclosed by Reinhart et al., 2002, Genes &Development 16:1616-1626, Llave et al., 2002, Plant Cell 14:1605-1619and Wu et al., 2006, Development, 133.4211. In general, an RNA precursoris constructed by obtaining the sequence of known RNA precursor for anmiRNA and replacing the miRNA sequences therein with the miRNA sequencesdirected to the target gene of interest. Methods for constructingprecursor miRNAs and miRNAs that can be used to alter the expression ofspecific target genes are known in the art. See, for example, McManus etal., 2002, RNA 8:842-850. Alternatively, precursor miRNAs from the sameor a different plant (or other) species can be isolated by methods knownin the art (Reinhart et al., 2002, Genes & Development 16:1616-1626 andLlave et al., 2002, Plant Cell 14:1605-1619).

The precursor miRNA can be cleaved or otherwise processed by the plantcell to produce miRNA substantially complementary to at least a portionof an mRNA sequence encoding a gene. For a target gene of interest, themiRNA, such as the miR167, is complementary or partially complementaryto a region of the target gene. That is the miRNA comprises a regionthat is completely complementary to a region of the target gene, or themiRNA comprises a region that is partially complementary to a region ofthe target gene. By partially complementary, it is intended thecorresponding regions of the target gene and the miRNA have one, two,three, or more mismatched bases. It is recognized in the art that miRNAsmay not be completely complementary to the region of a target gene.

The double-stranded portion of the nucleic acid may remaindouble-stranded even if the two nucleotide regions forming thedouble-stranded portions are not perfectly complementary to each other,i.e., the two regions are substantially complementary to each other. Forexample, additions, deletions, substitutions, etc. may occur in oneregion relative to the other, and in some cases, one region itself maycontain stem-loop motifs or other secondary structures that are notfound in the complementary region. However, the two regions may besubstantially complementary in that the two regions can interact in apredictable fashion to produce the double-stranded or “stem” portion ofthe stem-loop motif. Stem-loop motifs are well known in the art. Theactual primary sequence of nucleotides within the stem-loop structure isnot critical to the practice of the invention, as long as the secondarystructure is generally present. Those of ordinary skill in the art willbe able to determine, given a nucleic acid having a primary sequence ofnucleotides, whether the nucleic acid is able to form a stem-loop motif.

The precursor miRNA may include homologous or heterologous stem-loop andmiRNA sequence components. Transfection of a precursor miRNA containinga heterologous sequence into a cell may result in the formation of atransgenic plant cell. Thus, in some instances, the precursor miRNA,such as a precursor miR167, will include a stem-loop structure that isnot ordinarily associated in nature with the miRNA with which it isassociated in the precursor molecule. In a homologous structure the twocomponents are ordinarily found in association with one another innature. A heterologous precursor miRNA may be produced by replacing aportion (e.g., the homologous miRNA from the stem-loop structure) of aprecursor miRNA taken from a plant cell with a sequence substantiallycomplementary to another gene, for example, a gene that is desired to beinhibited or otherwise altered. The portion of the precursor miRNA thatis substantially complementary to the replaced miRNA portion may also bereplaced with a sequence that is substantially complementary to the genenewly added to the precursor miRNA. In some cases, a heterologousprecursor miRNA may be produced by selecting a sequence substantiallycomplementary to a gene that is desired to be inhibited or otherwisealtered, pairing it with a substantially complementary, and adding thepaired sequence to a stem-loop structure, which may be artificiallygenerated in some cases. For example, a precursor miRNA may be createdby selecting a sequence substantially complementary to a gene that isdesired to be inhibited or otherwise altered, pairing it with asubstantially complementary sequence, and adding a sequence thatincludes a stem-loop motif (other sequences may optionally be includedwithin the stem-loop motif as well, in some embodiments). Optionally,one or more other sequences may also be added to the precursor miRNA.

miRNAs, such as an miR167, that can be used in the compositions andmethods provided herein may be derived from any plant (or other)species, such as, for example, Arabidopsis thaliana or other Arabidopsisspecies, Oryza sativa or other Oryza species, or the like.

Precursor miRNA sequences are typically produced by transcribing aportion of the cell's DNA into RNA. Thus, a nucleotide sequence able tobe transcribed by a plant cell into precursor miRNA that is cleavable bythe plant cell to produce miRNA. The gene to be partially or totallyinhibited, or otherwise altered, may be any plant cell gene that iscapable of being transcribed into a protein. In certain embodiments,miR167 regulates, either directly or indirectly, expression of ARF8, andpotentially regulates, either directly or indirectly, expression of ARF6(At1g30330), or At3g61310, and/or downstream gene products modulated byARF8 (FIG. 2B), such as At3g61310, At1g76420, At1g24260, At1g79350,At1g63470, At2g20100, At3g45610; At2g26330, At3g57830, At2g01210;At3g16170, At1g48100, At1g11730, At1g70710 (CEL1), At1g32930, At3g13000,At2g38160, At1g03170, At3g13510, At2g23700, At3g11000, At3g10310,At2g42120, At1g15570, At2g26180, At1g67320 and/or At2g44440.

The particular gene to be inhibited will depend on the desired change tothe cell. The methods and compositions of the invention are not limitedto a particular gene. The nucleotide sequence may be isolated, e.g.,from plant cells, according to certain embodiments, and the nucleotidesequence may be either DNA or RNA. Those of ordinary skill in the artwill be able to determine if a given nucleotide sequence encodes aprecursor miRNA sequence. In some embodiments, as further discussedbelow, the nucleotide sequence may be delivered to a plant cell, such asa root cell (e.g., a pericycle cell) and then the nucleotide sequencemay then be expressed by the plant cell. In certain embodiments, thenucleotide sequence encodes a precursor miR167 operably linked to apericycle-specific or pericycle-preferred promoter, wherein the miR167is overexpressed in the plant as compared to a wild-type plant. In otherembodiments, the miR167 is inducibly-expressed in the plant.

Precursor miRNAs, according to the invention, are not limited towild-type or homologous precursor miRNAs. In some embodiments, amodified precursor miRNA, where a portion of the precursor miRNA, suchas the region encoding the mature miRNA, is replaced in some fashionwith another miRNA sequence. Any suitable miRNA sequence may be used,for example, miRNA sequences directed to the inhibition of a gene,partially or totally, within the plant cell. In some cases, the newmiRNA sequence added to the precursor miRNA may be shorter or longerthan the original miRNA sequence. For instance, one aspect of theinvention is generally directed to an isolated precursor miRNA able toinhibit a gene in a plant cell. A portion of a precursor miRNA, or anucleotide sequence able to be transcribed by a plant cell intoprecursor miRNA, may be replaced with a sequence substantiallycomplementary to a gene to be inhibited. Methods using such isolatedprecursor miRNA, or nucleotide sequences encoding such precursor miRNA,to partially or totally inhibit, or otherwise alter a gene are alsoprovided in certain embodiments. For instance, a precursor miRNA may beinserted into a plant cell, and/or a nucleotide sequence encoding aprecursor miRNA may be inserted into a plant cell such that thenucleotide sequence can be transcribed by the plant cell into precursormiRNA. In specific embodiments, the miRNA is miR167.

Thus, the present invention also provides, according to various aspects,methods and compositions for the expression of precursor miRNA inplants, for example, to inhibit a gene. In some cases, the expression ofmiRNA and/or precursor miRNA in a plant cell can also be altered byaltering the environment that the cell is in. In certain embodiments,the plant is in a nitrogen-moderate or nitrogen-rich environment.

In some embodiments, a precursor RNA construct is designed to produce atranscript that would be processed via the miRNA pathway to produce anmiRNA complementary to a target RNA, an RNA corresponding or transcribedby the target sequence. While not bound by any mechanism of action, themiRNAs alter the production, processing, stability, or translation ofthe target RNA and thereby alter the expression of the protein productof the targeted RNA. The miRNAs of the invention will be complementaryor substantially complementary to a target RNA that corresponds to thetarget gene of interest. In certain embodiments, the targetpolynucleotide is ARF8. By regulating ARF8, miR167 also indirectlyregulates downstream gene products modulated by ARF8 (FIG. 2B), such asAt3g61310, At1g76420, At1g24260, At1g79350, At1g63470, At2g20100,At3g45610; At2g26330, At3g57830, At2g01210; At3g16170, At1g48100,At1g11730, At1g70710 (CEL1), At1g32930, At3g13000, At2g38160, At1g03170,At3g13510, At2g23700, At3g11000, At3g10310, At2g42120, At1g15570,At2g26180, At1g67320 and/or At2g44440. miR167 is also predicted toregulate, either directly or indirectly, expression of ARF6 (At1g30330)and At3g61310.

The miRNA will generally be small molecules comprising about 15 to about30 nucleotides, about 20 to about 28 nucleotides, more specificallyabout 21-24 nucleotides. In certain embodiments, the miR167 is 24nucleotides in length. Generally the miRNA will be completelycomplementary to the target RNA, however, mismatches may be tolerated.Generally from 1-about 6 mismatches may occur, more specifically about2-3 mismatched nucleotides may be included in the miRNA. While themismatched nucleotides may occur throughout the miRNA sequence,preferably, they are located near the center of the molecule. In thismanner, an miRNA sequence can be designed to modulate the expression ofa target sequence. The miRNA is expressed as part of a precursor RNAconstruct. As noted above, once the precursor RNA construct is expressedin the plant cell, it is processed to produce the miRNAs, preferablymiR167.

6.2 Modulation of Gene Expression

The methods of the invention involve nitrogen-responsive miRNA, such asmiR167, modulation of the expression of one, two, or more targetnucleotide sequences in a plant, and preferably the plant pericycle, areprovided. That is, the expression of a target nucleotide sequence ofinterest (or downstream product thereof) may be increased or decreased.

The target nucleotide sequences may be endogenous or exogenous inorigin. By “modulate expression of a target gene” is intended that theexpression of the target gene is increased or decreased relative to theexpression level in a plant that has not been altered by the methodsdescribed herein. For example, in some embodiments, miR167 regulates,either directly or indirectly, expression of ARF8, and thus, in turn,downstream gene products modulated by ARF8 (FIG. 2B), such as At3g61310,At1g76420, At1g24260, At1g79350, At1g63470, At2g20100, At3g45610;At2g26330, At3g57830, At2g01210; At3g16170, At1g48100, At1g11730,At1g70710 (CEL1), At1g32930, At3g13000, At2g38160, At1g03170, At3g13510,At2g23700, At3g11000, At3g10310, At2g42120, At1g15570, At2g26180,At1g67320 and/or At2g44440. miR167 is also predicted to regulate, eitherdirectly or indirectly, expression of ARF6 (At1g30330) and At3g61310.

By “increased expression” is intended that expression of the targetnucleotide sequence is increased over expression observed inconventional transgenic lines for heterologous genes and over endogenouslevels of expression for homologous genes. Heterologous or exogenousgenes comprise genes that do not occur in the plant of interest in itsnative state. Homologous or endogenous genes are those that are nativelypresent in the plant genome. Generally, expression of the targetsequence is substantially increased. That is expression is increased atleast about 25%-50%, preferably about 50%-100%, more preferably about100%, 200% and greater.

By “decreased expression” is intended is intended that expression of thetarget nucleotide sequence is decreased below expression observed inconventional transgenic lines for heterologous genes and belowendogenous levels of expression for homologous genes. Generally,expression of the target nucleotide sequence of interest issubstantially decreased. That is expression is decreased at least about25%-50%, preferably about 50%-100%, more preferably about 100%, 200% andgreater.

Expression levels may be assessed by determining the level of a geneproduct by any method known in the art including, but not limited todetermining the levels of the RNA and protein encoded by a particulartarget gene. For genes that encode proteins, expression levels maydetermined, for example, by quantifying the amount of the proteinpresent in plant cells, or in a plant or any portion thereof.Alternatively, it desired target gene encodes a protein that has a knownmeasurable activity, then activity levels may be measured to assessexpression levels.

The target nucleotide sequence comprises any nucleotide sequence or geneof interest, including genes, regulatory sequences, and the like.Exemplary polynucleotides regulated either directly or indirectly bymiR167 include, but are not limited to, ARF8, ARF6, At3g61310,At1g76420, At1g24260, At1g79350, At1g63470, At2g20100, At3g45610;At2g26330, At3g57830, At2g01210; At3g16170, At1g48100, At1g11730,At1g70710 (CEL1), At1g32930, At3g13000, At2g38160, At1g03170, At3g13510,At2g23700, At3g11000, At3g10310, At2g42120, At1g15570, At2g26180,At1g67320 and/or At2g44440.

6.3 Modulators

The regulation of a gene via miRNA can be used in combination with amodulator. Such modulators include, but are not limited to, viral (orcellular) proteins that regulate miRNA accumulation. The modulators ofthe invention are capable of altering the levels of at least one miRNAin a plant. For example, HC-Pro, a viral suppressor of RNA silencing,enhances the accumulation of endogenous miRNAs. Thus, in certainembodiments, modulators can be used in combination with the miRNAprecursor constructs provided herein to enhance the regulatorycapabilities of miRNA, such as miR167, that correspond to targetsequences of interest, such as ARF8. In some embodiments, a modulator isused to alter regulation of the miRNA pathway. In certain embodiments,the modulator works to enhance the accumulation of miRNAs.

Variant modulator proteins can also be utilized in certain embodiments.Variant proteins encompassed by the present invention are biologicallyactive, that is they continue to possess the desired biological activityof the native protein, that is, modulator activity as described herein.Biologically active variants of a native modulator protein of theinvention will have at least about 40%, 50%, 60%, 65%, 70%, generally atleast about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or moresequence identity to the amino acid sequence for the native protein asdetermined by sequence alignment programs described elsewhere hereinusing default parameters. A biologically active variant of a protein ofthe invention may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

6.4 Transformation/Transfection

Any method or delivery system may be used for the delivery and/ortransfection of the precursor miRNA, such as miR167, or a nucleotidesequence able to be transcribed to produce precursor miRNA in the cell.The precursor miRNA, or the nucleotide sequence able to be transcribedto produce precursor miRNA, may be delivered to the plant cell eitheralone, or in combination with other agents.

Transfection may be accomplished by a wide variety of means, as is knownto those of ordinary skill in the art. Such methods include, but are notlimited to, Agrobacterium-mediated transformation (e.g., Komari et al.,1998, Curr. Opin. Plant Biol., 1:161), particle bombardment mediatedtransformation (e.g., Finer et al., 1999, Curr. Top. Microbiol.Immunol., 240:59), protoplast electroporation (e.g., Bates, 1999,Methods Mol. Biol., 111:359), viral infection (e.g., Porta andLomonossoff, 1996, Mol. Biotechnol. 5:209), microinjection, and liposomeinjection. Other exemplary delivery systems that can be used tofacilitate uptake by a cell of the nucleic acid include calciumphosphate and other chemical mediators of intracellular transport,microinjection compositions, and homologous recombination compositions(e.g., for integrating a gene into a preselected location within thechromosome of the cell). Alternative methods may involve, for example,the use of liposomes, electroporation, or chemicals that increase free(or “naked”) DNA uptake, transformation using viruses or pollen and theuse of microprojection. Standard molecular biology techniques are commonin the art (e.g., Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, New York). Forexample, in one embodiment of the present invention, Arabidopsis oranother plant species is transformed with a gene encoding a precursormiRNA such as miR167 using Agrobacterium.

One of skill in the art will be able to select an appropriate vector forintroducing the miR167-encoding nucleic acid sequence in a relativelyintact state. Thus, any vector which will produce a plant carrying theintroduced miR167-encoding nucleic acid should be sufficient. Theselection of the vector, or whether to use a vector, is typically guidedby the method of transformation selected.

The transformation of plants in accordance with the invention may becarried out in essentially any of the various ways known to thoseskilled in the art of plant molecular biology. (See, for example,Methods of Enzymology, Vol. 153, 1987, Wu and Grossman, Eds., AcademicPress, incorporated herein by reference).

Plant cells and plants can comprise two or more nucleotide sequenceconstructs. Any means for producing a plant comprising the nucleotidesequence constructs described herein are encompassed by the presentinvention. For example, a nucleotide sequence encoding the modulator canbe used to transform a plant at the same time as the nucleotide sequenceencoding the precursor RNA. The nucleotide sequence encoding theprecursor mRNA can be introduced into a plant that has already beentransformed with the modulator nucleotide sequence. Alternatively,transformed plants, one expressing the modulator and one expressing theRNA precursor, can be crossed to bring the genes together in the sameplant. Likewise, viral vectors may be used to express gene products byvarious methods generally known in the art. Suitable plant viral vectorsfor expressing genes should be self-replicating, capable of systemicinfection in a host, and stable. Additionally, the viruses should becapable of containing the nucleic acid sequences that are foreign to thenative virus forming the vector. Transient expression systems may alsobe used.

Agrobacterium transformation is widely used by those skilled in the artto transform dicotyledonous species. Recently, there has beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(Toriyarna et al., 1988, Bio/Technology 6:1072-1074; Zhang et al., 1988,Plant Cell Rep. 7:379-384; Zhang et al., 1988, Theor. Appl. Genet.76:835-840; Shimamoto et al., 1989, Nature 338:274-276; Datta et al.,1990, Bio/Technology 8: 736-740; Christou et al., 1991, Bio/Technology9:957-962; Peng et al., 1991, International Rice Research Institute,Manila, Philippines, pp. 563-574; Cao et al., 1992, Plant Cell Rep.11:585-591; Li et al., 1993, Plant Cell Rep. 12:250-255; Rathore et al.,1993, Plant Mol. Biol. 21:871-884; Fromm et al., 1990, Bio/Technology8:833-839; Tomes et al., 1995, “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,Berlin); D'Halluin et al., 1992, Plant Cell 4:1495-1505; Walters et al.,1992, Plant Mol. Biol. 18:189-200; Koziel et al., 1993, Biotechnology11: 194-200; Vasil, I. K., 1994, Plant Mol. Biol. 25:925-937; Weeks etal., 1993, Plant Physiol. 102:1077-1084; Somers et al., 1992,Bio/Technology 10: 1589-1594; WO 92/14828). In particular, Agrobacteriummediated transformation is now emerging also as an highly efficienttransformation method in monocots (Hiei et al., 1994, The Plant Journal6:271-282). See also, Shimamoto, K., 1994, Current Opinion inBiotechnology 5:158-162; Vasil et al., 1992, Bio/Technology 10:667-674;Vain et al., 1995, Biotechnology Advances 13 (4):653-671; Vasil et al.,1996, Nature Biotechnology 14:702).

The particular choice of a transformation technology will be determinedby its efficiency to transform certain plant species as well as theexperience and preference of the person practicing the invention with aparticular methodology of choice. It will be apparent to the skilledperson that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

6.4.1 Agrobacterium

miR167-encoding nucleic acid sequences utilized in the present inventioncan be introduced into plant cells using Ti plasmids of Agrobacteriumtumefaciens (A. tumefaciens), root-inducing (Ri) plasmids ofAgrobacterium rhizogenes (A. rhizogenes), and plant virus vectors. Forreviews of such techniques see, for example, Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, NY, SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9, and Horsch et al., 1985, Science,227:1229.

In using an A. tumefaciens culture as a transformation vehicle, it ismost advantageous to use a non-oncogenic strain of Agrobacterium as thevector carrier so that normal non-oncogenic differentiation of thetransformed tissues is possible. It is also preferred that theAgrobacterium harbor a binary Ti plasmid system. Such a binary systemcomprises 1) a first Ti plasmid having a virulence region essential forthe introduction of transfer DNA (T-DNA) into plants, and 2) a chimericplasmid. The chimeric plasmid contains at least one border region of theT-DNA region of a wild-type Ti plasmid flanking the nucleic acid to betransferred. Binary Ti plasmid systems have been shown effective in thetransformation of plant cells (De Framond, Biotechnology, 1983, 1:262;Hoekema et al., 1983, Nature, 303:179). Such a binary system ispreferred because it does not require integration into the Ti plasmid ofA. tumefaciens, which is an older methodology.

In some embodiments, a disarmed Ti-plasmid vector carried byAgrobacterium exploits its natural gene transferability (EP-A-270355,EP-A-01 16718, Townsend et al., 1984, NAR, 12:8711, U.S. Pat. No.5,563,055).

Methods involving the use of Agrobacterium in transformation accordingto the present invention include, but are not limited to: 1)co-cultivation of Agrobacterium with cultured isolated protoplasts; 2)transformation of plant cells or tissues with Agrobacterium; or 3)transformation of seeds, apices or meristems with Agrobacterium.

In addition, gene transfer can be accomplished by in plantatransformation by Agrobacterium, as described by Bechtold et al., (C.R.Acad. Sci. Paris, 1993, 316:1194). This approach is based on the vacuuminfiltration of a suspension of Agrobacterium cells.

In certain embodiments, a miR167-encoding nucleic acid is introducedinto plant cells by infecting such plant cells, an explant, a meristemor a seed, with transformed A. tumefaciens as described above. Underappropriate conditions known in the art, the transformed plant cells aregrown to form shoots, roots, and develop further into plants.

Other methods described herein, such as microprojectile bombardment,electroporation and direct DNA uptake can be used where Agrobacterium isinefficient or ineffective. Alternatively, a combination of differenttechniques may be employed to enhance the efficiency of thetransformation process, e.g., bombardment with Agrobacterium-coatedmicroparticles (EP-A-486234) or microprojectile bombardment to inducewounding followed by co-cultivation with Agrobacterium (EP-A-486233).

6.4.2 CaMV

In some embodiments, cauliflower mosaic virus (CaMV) is used as a vectorfor introducing miR167 nucleic acid into plant cells (U.S. Pat. No.4,407,956). CaMV viral DNA genome can be inserted into a parentbacterial plasmid creating a recombinant DNA molecule which can bepropagated in bacteria. After cloning, the recombinant plasmid again canbe cloned and further modified by introduction of the desired nucleicacid sequence. The modified viral portion of the recombinant plasmid canthen be excised from the parent bacterial plasmid, and used to inoculatethe plant cells or plants.

6.4.3 Mechanical and Chemical Means

In some embodiments, miR167-encoding nucleic acid is introduced into aplant cell using mechanical or chemical means. Exemplary mechanical andchemical means are provided below.

As used herein, the term “contacting” refers to any means of introducinga miR167-encoding nucleic acid into a plant cell, including chemical andphysical means as described above. Preferably, contacting refers tointroducing the nucleic acid or vector containing the nucleic acid intoplant cells (including an explant, a meristem or a seed), via A.tumefaciens transformed with the miR167-encoding nucleic acid asdescribed above.

6.4.3.1 Microinjection

In one embodiment, the miR167 nucleic acid can be mechanicallytransferred into the plant cell by microinjection using a micropipette.See, e.g., WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al.,1987, Plant Tissue and Cell Culture, Academic Press, Crossway et al.,1986, Biotechniques 4:320-334.

6.4.3.2 PEG

In other embodiment, the nucleic acid can also be transferred into theplant cell by using polyethylene glycol (PEG) which forms aprecipitation complex with genetic material that is taken up by thecell.

6.4.3.3 Electroporation

Electroporation can be used, in another set of embodiments, to deliver anucleic acid to the cell, e.g., precursor miRNA, or a nucleotidesequence able to be transcribed to produce precursor miRNA (see, e.g.,Fromm et al., 1985, PNA 5, 82:5824). “Electroporation,” as used herein,is the application of electricity to a cell, such as a plant protoplast,in such a way as to cause delivery of a nucleic acid into the cellwithout killing the cell. Typically, electroporation includes theapplication of one or more electrical voltage “pulses” having relativelyshort durations (usually less than 1 second, and often on the scale ofmilliseconds or microseconds) to a media containing the cells. Theelectrical pulses typically facilitate the non-lethal transport ofextracellular nucleic acids into the cells. The exact electroporationprotocols (such as the number of pulses, duration of pulses, pulsewaveforms, etc.), will depend on factors such as the cell type, the cellmedia, the number of cells, the substance(s) to be delivered, etc., andcan be determined by those of ordinary skill in the art. Electroporationis discussed in greater detail in, e.g., EP 290395, WO 8706614, Riggs etal., 1986, Proc. Natl. Acad. Sci. USA 83:5602-5606; D'Halluin et al.,1992, Plant Cell 4:1495-1505). Other forms of direct DNA uptake can alsobe used in the methods provided herein, such as those discussed in,e.g., DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611, Paszkowski etal., 1984, EMBO J. 3:2717-2722.

6.4.3.4 Ballistic and Particle Bombardment

Another method for introducing a miR167-encoding nucleic acid into aplant cell is high velocity ballistic penetration by small particleswith the nucleic acid to be introduced contained either within thematrix of such particles, or on the surface thereof (Klein et al., 1987,Nature 327:70). Genetic material can be introduced into a cell usingparticle gun (“gene gun”) technology, also called microprojectile ormicroparticle bombardment. In this method, small, high-density particles(microprojectiles) are accelerated to high velocity in conjunction witha larger, powder-fired macroprojectile in a particle gun apparatus. Themicroprojectiles have sufficient momentum to penetrate cell walls andmembranes, and can carry RNA or other nucleic acids into the interiorsof bombarded cells. It has been demonstrated that such microprojectilescan enter cells without causing death of the cells, and that they caneffectively deliver foreign genetic material into intact tissue.Bombardment transformation methods are also described in Sanford et al.(Techniques 3:3-16, 1991) and Klein et al. (Bio/Techniques 10:286,1992). Although, typically only a single introduction of a new nucleicacid sequence(s) is required, this method particularly provides formultiple introductions.

Particle or microprojectile bombardment are discussed in greater detailin, e.g., the following references: U.S. Pat. No. 5,100,792,EP-A-444882, EP-A-434616; Sanford et al., U.S. Pat. No. 4,945,050; Tomeset al., 1995, “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);and McCabe et al., 1988, Biotechnology 6:923-926.

6.4.3.5 Colloidal Dispersion

In other embodiments, a colloidal dispersion system may be used tofacilitate delivery of a nucleic acid into the cell, for example,precursor miRNA, or a nucleotide sequence able to be transcribed toproduce precursor miRNA. As used herein, a “colloidal dispersion system”refers to a natural or synthetic molecule, other than those derived frombacteriological or viral sources, capable of delivering to and releasingthe nucleic acid to the cell. Colloidal dispersion systems include, butare not limited to, macromolecular complexes, beads, and lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. One example of a colloidal dispersion system is a liposome.Liposomes are artificial membrane vessels. It has been shown that largeunilamellar vessels (“LUV”), which-range in size from 0.2 to 4.0microns, can encapsulate large macromolecules within the aqueousinterior and these macromolecules can be delivered to cells in abiologically active form (e.g., Fraley et al., 1981, Trends Biochem.Sci., 6:77).

6.4.3.6 Lipids

Lipid formulations for the transfection and/or intracellular delivery ofnucleic acids are commercially available, for instance, from QIAGEN, forexample as EFFECTENE® (a non-liposomal lipid with a special DNAcondensing enhancer) and SUPER-FECT® (a novel acting dendrimerictechnology) as well as Gibco BRL, for example, as LIPOFECTIN® andLIPOFECTACE®, which are formed of cationic lipids such asN-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (“DOTMA”)and dimethyl dioctadecylammonium bromide (“DDAB”). Liposomes are wellknown in the art and have been widely described in the literature, forexample, in Gregoriadis, G., 1985, Trends in Biotechnology 3:235-241;Freeman et al., 1984, Plant Cell Physiol. 29:1353).

6.4.3.7 Other Methods

In addition to the above, other physical methods for the transformationof plant cells are reviewed in the following and can be used in themethods provided herein. Oard, 1991, Biotech. Adv. 9:1-11. Seegenerally, Weissinger et al., 1988, sAnn. Rev. Genet. 22:421-477;Sanford et al., 1987, Particulate Science and Technology 5:27-37;Christou et al., 1988, Plant Physiol. 87:671-674; McCabe et al., 1988,Bio/Technology 6:923-926; Finer and McMullen, 1991, In vitro Cell Dev.Biol. 27P: 175-182; Singh et al., 1998, Theor. Appl. Genet. 96:319-324;Datta et al., 1990, Biotechnology 8:736-740; Klein et al., 1988, Proc.Natl. Acad. Sci. USA 85:4305-4309; Klein et al., 1988, Biotechnology6:559-563; Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat.Nos. 5,322,783 and 5,324,646; Klein et al., 1988, Plant Physiol.91:440-444; Fromm et al., 1990, Biotechnology 8:833-839; Hooykaas-VanSlogteren et al., 1984, Nature (London) 311:763-764; Bytebier et al.,1987, Proc. Natl. Acad. Sci. USA 84:5345-5349; De Wet et al., 1985, TheExperimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman,N.Y.), pp. 197-209; Kaeppler et al., 1990, Plant Cell Reports 9:415-418and Kaeppler et al., 1992, Theor. Appl. Genet. 84:560-566; Li et al.,1993, Plant Cell Reports 12:250-255 and Christou and Ford, 1995, Annalsof Botany 75:407-413; Osjoda et al., 1996, Nature Biotechnology14:745-750; all of which are herein incorporated by reference.

6.5 Nucleic Acid Constructs

The RNA precursor and modulator sequences of the invention may beprovided in nucleotide sequence constructs or expression cassettes forexpression in the plant of interest. The cassette will include 5′ and 3′regulatory sequences operably linked to an miRNA nucleotide sequence ormodulator nucleotide sequence of the invention.

The expression cassette may additionally contain at least one additionalgene to be cotransformed into the organism. Alternatively, theadditional gene(s) can be provided on multiple expression cassettes.

In certain embodiments, an expression cassette can be used with aplurality of restriction sites for insertion of the sequences of theinvention to be under the transcriptional regulation of the regulatoryregions. The expression cassette can additionally contain selectablemarker genes (see below).

The expression cassette will generally include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of the invention, and a transcriptional and translationaltermination region functional in plants. The transcriptional initiationregion, the promoter, may be native or analogous or foreign orheterologous to the plant host. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. By “foreign” isintended that the transcriptional initiation region is not found in thenative plant into which the transcriptional initiation region isintroduced. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See alsoGuerineau et al., 1991, Mol. Gen. Genet. 262:141-144; Proudfoot, 1991,Cell 64:671-674; Sanfacon et al., 1991, Genes Dev. 5:141-149; Mogen etal., 1990, Plant Cell 2:1261-1272; Munroe et al., 1990, Gene 91:151-158;Ballas et al., 1989, Nucleic Acids Res. 17:7891-7903; and Joshi et al.,1987, Nucleic Acid Res. 15:9627-9639.

In some embodiments, a nucleic acid (e.g., precursor miRNA, or anucleotide sequence able to be transcribed to produce precursor miRNA)can be delivered to the cell in a vector. As used herein, a “vector” isany vehicle capable of facilitating the transfer of the nucleic acid tothe cell such that the nucleic acid can be processed and/or expressed inthe cell. The vector may transport the nucleic acid to the cells withreduced degradation, relative to the extent of degradation that wouldresult in the absence of the vector. The vector optionally includes geneexpression sequences or other components (such as promoters and otherregulatory elements) able to enhance expression of the nucleic acidwithin the cell. The invention also encompasses the cells transfectedwith these vectors, including those cells previously described. Incertain embodiments, the cells are pericycle cells transfected ortransformed with a vector that specifically (or preferably)overexpresses miR167 in the pericycle cells of the plant, but not in themajority of other cell types of the plant.

To commence a transformation process in certain embodiments, it is firstnecessary to construct a suitable vector and properly introduce it intothe plant cell. Vector(s) employed in the present invention fortransformation of a plant cell include a miR167-encoding nucleic acidsequence operably associated with a promoter, such as apericycle-specific promoter. Details of the construction of vectorsutilized herein are known to those skilled in the art of plant geneticengineering.

In general, vectors useful in the invention include, but are not limitedto, plasmids, phagemids, viruses, other vehicles derived from viral orbacterial sources that have been manipulated by the insertion orincorporation of the nucleotide sequences (or precursor nucleotidesequences) of the invention. Viral vectors useful in certain embodimentsinclude, but are not limited to, nucleic acid sequences from thefollowing viruses: retroviruses; adenovirus, or other adeno-associatedviruses; mosaic viruses such as tobamoviruses; potyviruses, nepoviruses,and RNA viruses such as retroviruses. One can readily employ othervectors not named but known to the art. Some viral vectors can be basedon non-cytopathic eukaryotic viruses in which non-essential genes havebeen replaced with the nucleotide sequence of interest. Non-cytopathicviruses include retroviruses, the life cycle of which involves reversetranscription of genomic viral RNA into DNA with subsequent proviralintegration into host cellular DNA.

Genetically altered retroviral expression vectors can have generalutility for the high-efficiency transduction of nucleic acids. Standardprotocols for producing replication-deficient retroviruses (includingthe steps of incorporation of exogenous genetic material into a plasmid,transfection of a packaging cell lined with plasmid, production ofrecombinant retroviruses by the packaging cell line, collection of viralparticles from tissue culture media, and infection of the cells withviral particles) are well known to those of ordinary skill in the art.Examples of standard protocols can be found in Kriegler, M., 1990, GeneTransfer and Expression, A Laboratory Manual, W.H. Freeman Co., NewYork, or Murry, E. J. Ed., 1991, Methods in Molecular Biology, Vol. 7,Humana Press, Inc., Cliffton, N.J.

Another-example of a virus for certain applications is theadeno-associated virus, which is a double-stranded DNA virus. Theadeno-associated virus can be engineered to be replication-deficient andis capable of infecting a wide range of-cell types and species. Theadeno-associated virus further has advantages, such as heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages; and/or lack of superinfection inhibition, which may allowmultiple series of transductions.

Another vector suitable for use with the method provided herein is aplasmid vector. Plasmid vectors, have been extensively described in theart and are well-known to those of skill in the art. See, e.g., Sambrooket al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press. These plasmids may have a promotercompatible with the host cell, and the plasmids can express a peptidefrom a gene operatively encoded within the plasmid. Some commonly usedplasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript.Other plasmids are well-known to those of ordinary skill in the art.Additionally, plasmids may be custom-designed, for example, usingrestriction enzymes and ligation reactions, to remove and add specificfragments of DNA or other nucleic acids, as necessary. The presentinvention also includes vectors for producing nucleic acids or precursornucleic acids containing a desired nucleotide sequence (which can, forinstance, then be cleaved or otherwise processed within the cell toproduce a precursor miRNA). These vectors may include a sequenceencoding a nucleic acid and an in vivo expression element, as furtherdescribed below. In some cases, the in vivo expression element includesat least one promoter.

Where appropriate, the gene(s) for enhanced expression may be optimizedfor expression in the transformed plant. That is, the genes can besynthesized using plant-preferred codons corresponding to the plant ofinterest. Methods are available in the art for synthesizingplant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and5,436,391, and Murray et al., 1989, Nucleic Acids Res. 17:477-498.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whendesired, the sequence is modified to avoid predicted hairpin secondarymRNA structures. However, it is recognized that in the case ofnucleotide sequences encoding the miRNA precursors, one or more hairpinand other secondary structures may be desired for proper processing ofthe precursor into an mature miRNA and/or for the functional activity ofthe miRNA in gene silencing.

The expression cassettes can additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al., 1989,PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al., 1991, Nature 353:90-94);untranslated leader from the coat protein miRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al., 1987, Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al., 1989, Molecular Biology of RNA, ed.Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virusleader (MCMV) (Lommel et al., 1991, Virology 81:382-385). See also,Della-Cioppa et al., 1987, Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

6.6 Promoters and Other Regulatory Sequences

In the broad method of the invention, at least one nucleic acid sequenceencoding miR167 is operably linked with a promoter, such as apericyclel-preferred or pericycle-specific promoter. It may be desirableto introduce more than one copy of a miR167 polynucleotide into a plantfor enhanced miR167 expression. For example, multiple copies of a miR167polynucleotide would have the effect of increasing production of miR167even further in the plant. In specific embodiments, the miR167polynucleotide is expressed primarily or entirely in pericycle specificcells of the plant.

In general, promoters are found positioned 5′ (upstream) of the genesthat they control. Thus, in the construction of promoter genecombinations, the promoter is preferably positioned upstream of the geneand at a distance from the transcription start site that approximatesthe distance between the promoter and the gene it controls in thenatural setting. As is known in the art, some variation in this distancecan be tolerated without loss of promoter function. Similarly, thepreferred positioning of a regulatory element, such as an enhancer, withrespect to a heterologous gene placed under its control reflects itsnatural position relative to the structural gene it naturally regulates.In certain specific embodiments, the miR167 is under the control of apericycle specific promoter, and may optionally comprise otherregulatory elements that result in constitutive or inducible expressionof the miR167.

Thus, the nucleic acid, in one embodiment, is operably linked to a geneexpression sequence, which directs the expression of the nucleic acidwithin the cell (e.g., to produce the precursor miRNA). A “geneexpression sequence,” as used herein, is any regulatory nucleotidesequence, such as a promoter sequence or promoter-enhancer combination,which facilitates the efficient transcription and translation of thenucleotide sequence to which it is operably linked. The gene expressionsequence may, for example, be a eukaryotic promoter or a viral promoter,such as a constitutive or inducible promoter. Promoters and enhancersconsist of short arrays of DNA sequences that interact specifically withcellular proteins involved in transcription, for instance, as discussedin Maniatis et al., 1987, Science 236:1237. Promoter and enhancerelements have been isolated from a variety of eukaryotic sourcesincluding genes in plant, yeast, insect and mammalian cells and viruses(analogous control elements, i.e., promoters, are also found inprokaryotes). In some embodiments, the nucleic acid is linked to a geneexpression sequence which permits expression of the nucleic acid in aplant cell. A sequence which permits expression of the nucleic acid in aplant cell is one which is selectively active in the particular plantcell and thereby causes the expression of the nucleic acid in thesecells. Those of ordinary skill in the art will be able to easilyidentify promoters that are capable of expressing a nucleic acid in acell based on the type of plant cell.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. Generally, theRNA precursor nucleotide sequence and the modulator sequences can becombined with promoters of choice to alter gene expression if the targetsequences in the tissue or organ of choice. Thus, the RNA precursornucleotide sequence or modulator nucleotide sequence can be combinedwith constitutive, tissue-preferred, inducible, developmental, or otherpromoters for expression in plants depending upon the desired outcome.

The selection of a particular promoter and enhancer depends on what celltype is to be used and the mode of delivery. For example, a wide varietyof promoters have been isolated from plants and animals, which arefunctional not only in the cellular source of the promoter, but also innumerous other plant species. There are also other promoters (e.g.,viral and Ti-plasmid) which can be used. For example, these promotersinclude promoters from the Ti-plasmid, such as the octopine synthasepromoter, the nopaline synthase promoter, the mannopine synthasepromoter, and promoters from other open reading frames in the T-DNA,such as ORF7, etc. Promoters isolated from plant viruses include the 35Spromoter from cauliflower mosaic virus. Promoters that have beenisolated and reported for use in plants include ribulose-1,3-biphosphatecarboxylase small subunit promoter, phaseolin promoter, etc. Thus, avariety of promoters and regulatory elements may be used in theexpression vectors of the present invention.

Promoters useful in the compositions and methods provided herein includeboth natural constitutive and inducible promoters as well as engineeredpromoters. The CaMV promoters are examples of constitutive promoters.Other constitutive mammalian promoters include, but are not limited to,polymerase promoters as well as the promoters for the following genes:hypoxanthine phosphoribosyl transferase (“HPTR”), adenosine deaminase,pyruvate kinase, and alpha-actin.

Promoters useful as expression elements of the invention also includeinducible promoters. Inducible promoters are expressed in the presenceof an inducing agent. For example, a metallothionein promoter can beinduced to promote transcription in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart. The in vivo expression element can include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription, and can optionally include enhancersequences or upstream activator sequences.

For example, in some embodiments an inducible promoter is used to allowcontrol of nucleic acid expression through the presentation of externalstimuli (e.g., environmentally inducible promoters), as discussed below.Thus, the timing and amount of nucleic acid expression can be controlledin some cases. Non-limiting examples of expression systems, promoters,inducible promoters, environmentally inducible promoters, and enhancersare well known to those of ordinary skill in the art. Examples includethose described in International Patent Application Publications WO00/12714, WO 00/11175, WO 00/12713, WO 00/03012, WO 00/03017, WO00/01832, WO 99/50428, WO 99/46976 and U.S. Pat. Nos. 6,028,250,5,959,176, 5,907,086, 5,898,096, 5,824,857, 5,744,334, 5,689,044, and5,612,472. A general descriptions of plant expression vectors andreporter genes can also be found in Gruber et al., 1993, “Vectors forPlant Transformation,” in Methods in Plant Molecular Biology &Biotechnology, Glich et al., Eds., p. 89-119, CRC Press.

For plant expression vectors, viral promoters that can be used incertain embodiments include the 35S RNA and 19S RNA promoters of CaMV(Brisson et al., Nature, 1984, 310:511; Odell et al., Nature, 1985,313:810); the full-length transcript promoter from Figwort Mosaic Virus(FMV) (Gowda et al., 1989, J. Cell Biochem., 13D: 301) and the coatprotein promoter to TMV (Takamatsu et al., 1987, EMBO J. 6:307).Alternatively, plant promoters such as the light-inducible promoter fromthe small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO)(Coruzzi et al., 1984, EMBO J., 3:1671; Broglie et al., 1984, Science,224:838); mannopine synthase promoter (Velten et al., 1984, EMBO J.,3:2723) nopaline synthase (NOS) and octopine synthase (OCS) promoters(carried on tumor-inducing plasmids of Agrobacterium tumefaciens) orheat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley etal., 1986, Mol. Cell. Biol., 6:559; Severin et al., 1990, Plant Mol.Biol., 15:827) may be used. Exemplary viral promoters which functionconstitutively in eukaryotic cells include, for example, promoters fromthe simian virus, papilloma virus, adenovirus, human immunodeficiencyvirus, Rous sarcoma virus, cytomegalovirus, the long terminal repeats ofMoloney leukemia virus and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art.

To be most useful, an inducible promoter should 1) provide lowexpression in the absence of the inducer; 2) provide high expression inthe presence of the inducer; 3) use an induction scheme that does notinterfere with the normal physiology of the plant; and 4) have no effecton the expression of other genes. Examples of inducible promoters usefulin plants include those induced by chemical means, such as the yeastmetallothionein promoter which is activated by copper ions (Mett et al.,Proc. Natl. Acad. Sci., U.S.A., 90:4567, 1993); In2-1 and In2-2regulator sequences which are activated by substitutedbenzenesulfonamides, e.g., herbicide safeners (Hershey et al., PlantMol. Biol., 17:679, 1991); and the GRE regulatory sequences which areinduced by glucocorticoids (Schena et al., Proc. Natl. Acad. Sci.,U.S.A., 88:10421, 1991). Other promoters, both constitutive andinducible will be known to those of skill in the art.

A number of inducible promoters are known in the art. For resistancegenes, a pathogen-inducible promoter can be utilized. Such promotersinclude those from pathogenesis-related proteins (PR proteins), whichare induced following infection by a pathogen; e.g., PR proteins, SARproteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfiet al., 1983, Neth. J. Plant Pathol. 89:245-254; Uknes et al., 1992,Plant Cell 4:645-656; and Van Loon, 1985, Plant Mol. Virol. 4:111-116.Of particular interest are promoters that are expressed locally at ornear the site of pathogen infection. See, for example, Marineau et al.,1987, Plant Mol. Biol. 9:335-342; Matton et al., 1989, MolecularPlant-Microbe Interactions 2:325-331; Somsisch et al., 1986, Proc. Natl.Acad. Sci. USA 83:2427-2430; Somsisch et al., 1988, Mol. Gen. Genet.2:93-98; and Yang, 1996, Proc. Natl. Acad. Sci. USA 93:14972-14977. Seealso, Chen et al., 1996, Plant J. 10:955-966; Zhang et al., 1994, Proc.Natl. Acad. Sci. USA 91:2507-2511; Warner et al., 1993, Plant J.3:191-201; Siebertz et al., 1989, Plant Cell 1:961-968; U.S. Pat. No.5,750,386; Cordero et al., 1992, Physiol. Mol. Plant. Path. 41:189-200;and the references cited therein.

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in the DNAconstructs of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan, 1990, Ann. Rev.Phytopath. 28:425-449; Duan et al., 1996, Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al., 1989, Mol. Gen. Genet. 215:200-208); systemin (McGurlet al., 1992, Science 225:1570-1573); WIPI (Rohmeier et al., 1993, PlantMol. Biol. 22:783-792; Eckelkamp et al., 1993, FEBS Letters 323:73-76);MPI gene (Corderok et al., 1994, Plant J. 6 (2):141-150); and the like.Such references are herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1 a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al., 1991, Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al., 1998, Plant J. 14 (2):247-257) andtetramiR167e-inducible and tetramiR167e-repressible promoters (see, forexample, Gatz et al., 1991, Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Where enhanced expression in particular tissues is desired,tissue-preferred promoters can be utilized. Tissue-preferred promotersinclude those described by Yamamoto et al., 1997, Plant J. 12(2):255-265; Kawamata et al., 1997, Plant Cell Physiol. 38 (7):792-803;Hansen et al., 1997, Mol. Gen. Genet. 254 (3):337-343; Russell et al.,1997, Transgenic Res. 6 (2):157-168; Rinehart et al., 1996, PlantPhysiol. 112 (3):1331-1341; Van Camp et al., 1996, Plant Physiol. 112(2):525-535; Canevascini et al., 1996, Plant Physiol. 12 (2):513-524;Yamamoto et al., 1994, Plant Cell Physiol. 35 (5):773-778; Lam, 1994,Results Probl. Cell Differ. 20:181-196; Orozco et al., 1993, Plant Mol.Biol. 23 (6): 1129-1138; Matsuoka et al., 1993, Proc Natl. Acad. Sci.USA 90 (20):9586-9590; and Guevara-Garcia et al., 1993, Plant J 4(3):495-505.

The particular promoter selected should be capable of causing sufficientexpression to result in the production of an effective amount ofstructural gene product in the transgenic plant, e.g., miR167 to causedownregulation of genes such as ARF8, and increased lateral root growth,root biomass, overall plant growth or yield, and/or other phenotypesdescribed herein, such as when the plant is grown in the presence ofnitrogen (e.g., nitrogen-moderate or nitrogen-rich conditions) ascompared to wild type. The promoters used in the vector constructs ofthe present invention may be modified, if desired, to affect theircontrol characteristics. In certain embodiments, chimeric promoters canbe used.

There are promoters known which limit expression to particular plantparts or in response to particular stimuli. For example, a root specificpromoter would be desirable to obtain expression of miRNA in the plantroots, such as the pericycle. One skilled in the art will know of manysuch plant part-specific promoters which would be useful in the presentinvention. In certain embodiments, to provide pericycle-specificexpression, any of a number of promoters from genes in Arabidopsis canbe used. In some embodiments, the promoter from one (or more) of thefollowing genes may be used: (i) At1g1080, (ii) At3g60160, (iii)At1g24575, (iv) At3g45160, or (v) At1g23130. In specific embodiments, wewill also use (vi) promoter elements from the GFP-marker line used inGifford et al. (in preparation) (see also, Bonke et al., 2003, Nature426, 181-6; Tian et al., 2004, Plant Physiol 135, 25-38). Several of thepredicted genes have a number of potential orthologs in rice and poplarand thus we predict that they will be applicable for use in cropspecies; (i) Os04g44410, Os10g39560, Os06g51370, Os02g42310, Os01g22980,Os05g06660, and Poptr1#568263, Poptr1#555534, Poptr1#365170; (ii)Os04g49900, Os04g49890, Os01g67580, and Poptr1#87573, Poptr1#80582,Poptr1#565079, Poptr1#99223.

Promoters used in the nucleic acid constructs of the present inventioncan be modified, if desired, to affect their control characteristics.For example, the CaMV 35S promoter may be ligated to the portion of thessRUBISCO gene that represses the expression of ssRUBISCO in the absenceof light, to create a promoter which is active in leaves but not inroots. The resulting chimeric promoter may be used as described herein.For purposes of this description, the phrase “CaMV 35S” promoter thusincludes variations of CaMV 35S promoter, e.g., promoters derived bymeans of ligation with operator regions, random or controlledmutagenesis, etc. Furthermore, the promoters may be altered to containmultiple “enhancer sequences” to assist in elevating gene expression.

An efficient plant promoter that may be used in specific embodiments isan “overproducing” or “overexpressing” plant promoter. Overexpressingplant promoters that can be used in the compositions and methodsprovided herein include the promoter of the small sub-unit (“ss”) of theribulose-1,5-biphosphate carboxylase from soybean (e.g., Berry-Lowe etal., 1982, J. Molecular & App. Genet., 1:483), and the promoter of thechlorophyll a-b binding protein. These two promoters are known to belight-induced in eukaryotic plant cells. For example, see Cashmore,Genetic Engineering of plants: An Agricultural Perspective, p. 29-38;Coruzzi et al., 1983, J. Biol. Chem., 258:1399; and Dunsmuir et al.,1983, J. Molecular & App. Genet., 2:285.

The promoters and control elements of, e.g., SUCS (root nodules;broadbean; Kuster et al., 1993, Mol Plant Microbe Interact 6:507-14) forroots can be used in compositions and methods provided herein to confertissue specificity.

In certain embodiment, two promoter elements can be used in combination,such as, for example, (i) an inducible element responsive to a treatmentthat can be provided to the plant prior to N-fertilizer treatment, and(ii) a pericycle-specific expression element to drive miR167 expressionin the pericycle root cell type alone.

Any promoter of other expression element described herein or known inthe art may be used either alone or in combination with any otherpromoter or other expression element described herein or known in theart. For example, promoter elements that confer tissue specificexpression of a gene (e.g., miR167) can be used with other promoterelements conferring constitutive or inducible expression. In certainembodiments, two or more promoter elements can be used in combination,such as, for example, (i) an inducible element responsive to a treatmentthat can be provided to the plant prior to N-fertilizer treatment, and(ii) a pericycle-specific expression element to drive miR167 expressionin the pericycle root cell type alone.

6.7 Isolating Related Promoter Sequences

Promoter and promoter control elements that are related to thosedescribed in herein can also be used in the compositions and methodsprovided herein. Such related sequence can be isolated utilizing (a)nucleotide sequence identity; (b) coding sequence identity of related,orthologous genes; or (c) common function or gene products.

Relatives can include both naturally occurring promoters and non-naturalpromoter sequences. Non-natural related promoters include nucleotidesubstitutions, insertions or deletions of naturally-occurring promotersequences that do not substantially affect transcription modulationactivity. For example, the binding of relevant DNA binding proteins canstill occur with the non-natural promoter sequences and promoter controlelements of the present invention.

According to current knowledge, promoter sequences and promoter controlelements exist as functionally important regions, such as proteinbinding sites, and spacer regions. These spacer regions are apparentlyrequired for proper positioning of the protein binding sites. Thus,nucleotide substitutions, insertions and deletions can be tolerated inthese spacer regions to a certain degree without loss of function.

In contrast, less variation is permissible in the functionally importantregions, since changes in the sequence can interfere with proteinbinding. Nonetheless, some variation in the functionally importantregions is permissible so long as function is conserved.

The effects of substitutions, insertions and deletions to the promotersequences or promoter control elements may be to increase or decreasethe binding of relevant DNA binding proteins to modulate transcriptlevels of a polynucleotide to be transcribed. Effects may includetissue-specific or condition-specific modulation of transcript levels ofthe polypeptide to be transcribed. Polynucleotides representing changesto the nucleotide sequence of the DNA-protein contact region byinsertion of additional nucleotides, changes to identity of relevantnucleotides, including use of chemically-modified bases, or deletion ofone or more nucleotides are considered encompassed by the presentinvention.

Typically, related promoters exhibit at least 80% sequence identity,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%, even more preferably, at least 96%, at least97%, at least 98% or at least 99% sequence identity compared to thoseshown in Table 1. Such sequence identity can be calculated by thealgorithms and computers programs described above.

Usually, such sequence identity is exhibited in an alignment region thatis at least 75% of the length of a sequence or corresponding full-lengthsequence of a promoter described herein; more usually at least 80%; moreusually, at least 85%, more usually at least 90%, and most usually atleast 95%, even more usually, at least 96%, at least 97%, at least 98%or at least 99% of the length of a sequence of a promoter describedherein.

The percentage of the alignment length is calculated by counting thenumber of residues of the sequence in region of strongest alignment,e.g., a continuous region of the sequence that contains the greatestnumber of residues that are identical to the residues between twosequences that are being aligned. The number of residues in the regionof strongest alignment is divided by the total residue length of asequence of a promoter described herein. These related promoters mayexhibit similar preferential transcription as those promoters describedherein.

In certain embodiments, a promoter, such as a root-preferred or rootspecific promoter, can be identified by sequence homology or sequenceidentity to any root specific promoter identified herein. In otherembodiments, orthologous genes identified herein as root-specific genes(e.g., the same gene or different gene that if functionally equivalent)for a given species can be identified and the associated promoter canalso be used in the compostions and methods provided herein. Forexample, using high, medium or low stringency conditions, standardpromoter rules can be used to identify other useful promoters fromorthologous genes for use in the compositions and methods providedherein. In specific embodiments, the orthologous gene is a geneexpressed only or primarily in the root, such as pericycle cells. Insome embodiments, an expression vector that can be used in thecompositions and methods of the invention comprises a miR167polynucleotide operably linked to a regulatory nucleic acid sequencecontrolling the expression of a root specific or root preferred gene ofa same or different species of plant.

Polynucleotides can be tested for activity by cloning the sequence intoan appropriate vector, transforming plants with the construct andassaying for marker gene expression. Recombinant DNA constructs can beprepared, which comprise the polynucleotide sequences of the inventioninserted into a vector suitable for transformation of plant cells. Theconstruct can be made using standard recombinant DNA techniques(Sambrook et al., 1989) and can be introduced to the species of interestby Agrobacterium-mediated transformation or by other means oftransformation as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by (a) BAC: Shizuya et al., 1992, Proc.Natl. Acad. Sci. USA 89: 8794-8797; Hamilton et al., 1996, Proc. Natl.Acad. Sci. USA 93: 9975-9979; (b) YAC: Burke et al., 1987, Science236:806-812; (c) PAC: Stemberg N. et al., 1990, Proc Natl Acad Sci USA.January; 87 (1):103-7; (d) Bacteria-Yeast Shuttle Vectors: Bradshaw etal., 1995, Nucl Acids Res 23: 4850-4856; (e) Lambda Phage Vectors:Replacement Vector, e.g., Frischauf et al., 1983, J. Mol. Biol. 170:827-842; or Insertion vector, e.g., Huynh et al., 1985, In: Glover N M(ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press; T-DNAgene fusion vectors: Walden et al., 1990, Mol Cell Biol 1: 175-194; and(g) Plasmid vectors: Sambrook et al., infra.

Typically, the construct comprises a vector containing a sequence of thepresent invention operationally linked to any marker gene. Thepolynucleotide was identified as a promoter by the expression of themarker gene. Although many marker genes can be used, Green FluorescentProtein (GFP) is preferred. The vector may also comprise a marker genethat confers a selectable phenotype on plant cells. The marker mayencode biocide resistance, particularly antibiotic resistance, such asresistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosulfuron or phosphinotricin (seebelow). Vectors can also include origins of replication, scaffoldattachment regions (SARs), markers, homologous sequences, introns, etc.

6.8 Root Preferential Transcription

The invention also provides a method of providing increasedtranscription of a nucleic acid sequence in a selected tissue, such asthe root (e.g., pericycle cells of the root). The method comprisesgrowing a plant having integrated in its genome a nucleic acid constructcomprising, an exogeneous gene encoding a miR167, said gene operablyassociated with a tissue specific promoter, whereby transcription ofsaid gene is increased in said selected tissue.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas roots. Exemplary promoters include the root cdc2a promoter (Doerner,P. et al., 1996, Nature 380:520-523) or the root peroxidase promoterfrom wheat (Hertig, C. et al., 1991, Plant Mol. Biol. 16:171-174). Theoperation of a promoter may also vary depending on its location in thegenome. Thus, an inducible promoter may become fully or partiallyconstitutive in certain locations.

Specific promoters may be used in the compositions and methods providedherein. As used herein, “specific promoters” refers to a subset ofpromoters that have a high preference for modulating transcript levelsin a specific tissue or organ or cell and/or at a specific time duringdevelopment of an organism. By “high preference” is meant at least3-fold, preferably 5-fold, more preferably at least 10-fold still morepreferably at least 20-fold, 50-fold or 100-fold increase in transcriptlevels under the specific condition over the transcription under anyother reference condition considered. Typical examples of temporaland/or tissue or organ specific promoters of plant origin that can beused in the compositions and methods of the present invention, includeRCc2 and RCc3, promoters that direct root-specific gene transcription inrice (Xu et al., 1995, Plant Mol. Biol. 27:237 and TobRB27, aroot-specific promoter from tobacco (Yamamoto et al., 1991, Plant Cell3:371). Examples of tissue-specific promoters under developmentalcontrol include promoters that initiate transcription only in certaintissues or organs, such as roots

“Preferential transcription” is defined as transcription that occurs ina particular pattern of cell types or developmental times or in responseto specific stimuli or combination thereof. Non-limitive examples ofpreferential transcription include: high transcript levels of a desiredsequence in root tissues; detectable transcript levels of a desiredsequence in certain cell types during embryogenesis; and low transcriptlevels of a desired sequence under drought conditions. Such preferentialtranscription can be determined by measuring initiation, rate, and/orlevels of transcription.

Promoters and control elements providing preferential transcription in aroot can modulate growth, metabolism, development, nutrient uptake,nitrogen fixation, or modulate energy and nutrient utilization in hostcells or organisms. In a plant, for example, preferential modulation ofgenes, transcripts, and/or in a leaf, is useful (1) to modulate rootsize, shape, and development; (2) to modulate the number of roots, orroot hairs; (3) to modulate mineral, fertilizer, or water uptake; (4) tomodulate transport of nutrients; or (4) to modulate energy or nutrientusage in relation to other organs and tissues. Up-regulation andtranscription down-regulation is useful for these applications. Forinstance, genes, transcripts, and/or polypeptides that increase growth,for example, may require up-regulation of transcription. In contrast,transcriptional down-regulation may be desired to inhibit nutrient usagein a root to be directed to the leaf instead, for instance.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues, or organs of a root, produce transcriptlevels that are statistically significant as compared to other cells,organs or tissues. For preferential up-regulation of transcription,promoter and control elements produce transcript levels that are abovebackground of the assay.

Root-preferred promoters are known and can be selected from the manyavailable from the literature. See, for example, Hire et al., 1992,Plant Mol. Biol. 20 (2): 207-218 (soybean root-preferred glutaminesynthetase gene); Keller and Baumgartner, 1991, Plant Cell 3(10):1051-1061 (root-preferred control element in the GRP 1.8 gene ofFrench bean); Sanger et al., 1990, Plant Mol. Biol. 14 (3):433-443(root-preferred promoter of the mannopine synthase (MAS) gene ofAgrobacterium tumefaciens); Miao et al., 1991, Plant Cell 3 (1):11-22(full-length cDNA clone encoding cytosolic glutamine synthetase (GS),which is expressed in roots and root nodules of soybean). Bogusz et al.,1990, Plant Cell 2 (7):633-641 (root-preferred promoters from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa). Leach andAoyagi, 1991, Plant Science (Limerick) 79 (1):69-76 (ro1C and ro1Droot-inducing genes of Agrobacterium rhizogenes); Teeri et al., 1989,EMBO J. 8 (2):343-350) (octopine synthase and TR2′ gene); (VfENOD-GRP3gene promoter); Kuster et al., 1995, Plant Mol. Biol. 29 (4):759-772 andCapana et al., 1994, Plant Mol. Biol. 25 (4):681-691 ro1B promoter. Seealso U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;5,401,836; 5,110,732; and 5,023,179, root-specific glutamine synthetase(see Tingey et al., 1987, EMBO J., 6:1-9; Edwards et al., 1990, PNAS,87:3439-3463). In addition, promoters of the above-listed orthologousgenes in other plant species can be identified and used in thecompositions and methods provided herein.

In specific embodiments, the compositions and methods provided hereinuse root- or pericycle-specific promoters operably associated to anucleotide encoding miR167. In certain embodiments, the promoter is aconstitutive or inducible promoter.

6.9 Selectable Markers

Using any gene transfer technique, such as the above-listed techniques,an expression vector harboring the nucleic acid may be transformed intoa cell to achieve temporary or prolonged expression. Any suitableexpression system may be used, so long as it is capable of undergoingtransformation and expressing of the precursor nucleic acid in the cell.In one embodiment, a pET vector (Novagen, Madison, Wis.), or a pBIvector (Clontech, Palo Alto, Calif.) is used as the expression vector.In some embodiments an expression vector further encoding a greenfluorescent protein (“GFP”) is used to allow simple selection oftransfected cells and to monitor expression levels. Non-limitingexamples of such vectors include Clontech's “Living Colors Vectors”pEYFP and pEYFP-C.

The recombinant construct of the present invention may include aselectable marker for propagation of the construct. For example, aconstruct to be propagated in bacteria preferably contains an antibioticresistance gene, such as one that confers resistance to kanamycin,tetracycline, streptomycin, or chloramphenicol. Suitable vectors forpropagating the construct include plasmids, cosmids, bacteriophages orviruses, to name but a few.

In addition, the recombinant constructs may include plant-expressibleselectable or screenable marker genes for isolating, identifying ortracking of plant cells transformed by these constructs. Selectablemarkers include, but are not limited to, genes that confer antibioticresistances (e.g., resistance to kanamycin or hygromycin) or herbicideresistance (e.g., resistance to sulfonylurea, phosphinothricin, orglyphosate). Screenable markers include, but are not limited to, thegenes encoding .beta.-glucuronidase (Jefferson, 1987, Plant Molec Biol.Rep 5:387-405), luciferase (Ow et al., 1986, Science 234:856-859), B andC1 gene products that regulate anthocyanin pigment production (Goff etal., 1990, EMBO J 9:2517-2522).

In some cases, a selectable marker may be included with the nucleic acidbeing delivered to the cell. As used herein, the term “selectablemarker” refers to the use of a gene that encodes an enzymatic or otherdetectable activity (e.g., luminescence or fluorescence) that confersthe ability to grow in medium lacking what would otherwise be anessential nutrient. A selectable marker may also confer resistance to anantibiotic or drug upon the cell in which the selectable marker isexpressed. Selectable markers may be “dominant” in some cases; adominant selectable marker encodes an enzymatic or other activity (e.g.,luminescence or fluorescence) that can be detected in any cell or cellline.

Optionally, a selectable marker may be associated with themiR167-encoding nucleic acid. Preferably, the marker gene is anantibiotic resistance gene whereby the appropriate antibiotic can beused to select for transformed cells from among cells that are nottransformed. Examples of suitable selectable markers include adenosinedeaminase, dihydrofolate reductase, hygromycin-B-phosphotransferase,thymidine kinase, xanthine-guanine phospho-ribosyltransferase andamino-glycoside 3′-O-phosphotransferase II. Other suitable markers willbe known to those of skill in the art.

6.10 Selection and Identification of Transformed Plants and Plant Cells

According to the present invention, desired plants may be obtained byengineering the disclosed gene constructs into a variety of plant celltypes, including but not limited to, protoplasts, tissue culture cells,tissue and organ explants, pollens, embryos as well as whole plants. Inspecific embodiments, the miR167 gene constructs are engineered intoplant roots, such as pericycle cell, preferably with the use of apericycle specific promoter.

In an embodiment of the present invention, the engineered plant materialis selected or screened for transformants (those that have incorporatedor integrated the introduced gene construct(s)) following the approachesand methods described below. An isolated transformant may then beregenerated into a plant. Alternatively, the engineered plant materialmay be regenerated into a plant or plantlet before subjecting thederived plant or plantlet to selection or screening for the marker genetraits. Procedures for regenerating plants from plant cells, tissues ororgans, either before or after selecting or screening for markergene(s), are well known to those skilled in the art.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection may be performed by growing the engineered plantmaterial on media containing inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed plants and plant cells may also be identified byscreening for the activities of any visible marker genes (e.g., theβ-glucuronidase, luciferase, B or C1 genes) that may be present on therecombinant nucleic acid constructs of the present invention. Suchselection and screening methodologies are well known to those skilled inthe art.

Physical and biochemical methods also may be also to identify plant orplant cell transformants containing the gene constructs of the presentinvention. These methods include but are not limited to: 1) Southernanalysis or PCR amplification for detecting and determining thestructure of the recombinant DNA insert; 2) Northern blot, S1 RNaseprotection, primer-extension or reverse transcriptase-PCR amplificationfor detecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, Western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins. Additional techniques, such as in situ hybridization, enzymestaining, and immunostaining, also may be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the art.

miRNA167-transgenic plants may also be identified by examining thechange in expression of certain miRNA167-responsive genes. For example,a transgenic plant of the present invention can be identified bydetermining the expression of one or more of the genes listed inTable 1. In a plant of the invention, the expression of these genes isdown-regulated compared to wild-type.

6.11 Screening of Transformed Plants for Those with Improved AgronomicTraits

According to the present invention, to obtain plants with improvedagronomic characteristics, the transformed plants may be screened forthose exhibiting the desired physiological alteration. Alternatively,the transformed plants may be directly screened for those exhibiting thedesired agronomic changes. A plant with the desired improvement can beisolated by screening the engineered plants for altered expressionpattern or level of the miR167 (or precursor thereof) and/or expressionpattern or level of a direct or indirect target polynucleotide of themiR167, such as ARF8, or downstream gene products modulated by ARF8(FIG. 2B), such as At3g61310, At1g76420, At1g24260, At1g79350,At1g63470, At2g20100, At3g45610; At2g26330, At3g57830, At2g01210;At3g16170, At1g48100, At1g11730, At1g70710 (CEL1), At1g32930, At3g13000,At2g38160, At1g03170, At3g13510, At2g23700, At3g11000, At3g10310,At2g42120, At1g15570, At2g26180, At1g67320 and/or At2g44440. miR167 alsopotentially regulates, either directly or indirectly, expression of ARF6(At1g30330), or At3g61310. A plant can also be screened for lateral rootgrowth, root surface area, root biomass, nutrient uptake, overallincreased plant growth rate, enhanced vegetative yield, or improvedreproductive yields. The screening of the engineered plants can involveSouthern analysis to confirm the presence and number of transgeneinsertions; Northern analysis, RNase protection, primer extension,reverse transcriptase/PCR and the like to measure mRNA levels; measuringthe amino acid composition, free amino acid pool or total nitrogencontent of various plant tissues; monitoring numbers and types oflateral root primordia and lateral roots; measuring growth rates interms of fresh weight gains over time; or measuring plant yield in termsof total dry weight and/or total seed weight, or a combination of any ofthe above methods. The procedures and methods for examining theseparameters are well known to those skilled in the art.

In other embodiments, the screening of the transformed plants may be forimproved agronomic characteristics (e.g., faster growth, greatervegetative or reproductive yields, or improved protein contents, etc.),as compared to unengineered progenitor plants, when cultivated undernitrogen rich (e.g., N-rich soils or soil that has been fertilized withcommercial or organic fertilizer) growth conditions (i.e., cultivatedusing soils or media containing or receiving sufficient amounts ofnitrogen nutrients to sustain healthy plant growth).

Plants exhibiting increased growth and/or yield as compared withwild-type plants can be selected by visual observation, methods providedin the Examples, or other methods known in the art.

In another embodiment, the invention provides a method of producing aplant characterized as having increased growth and yield by contacting aplant capable of increased yield with a miR167-inducing amount of anagent which induces miR167 gene expression. Induction of miR167 geneexpression results in production of a plant having increased lateralroot growth and/or yield as compared to a plant not contacted with theagent.

A “plant capable of increased yield” refers to a plant that can beinduced to express its endogenous miR167 gene to achieve increasedyield. The term “promoter inducing amount” refers to that amount of anagent necessary to elevate miR167 gene expression above miR167expression in a plant cell not contacted with the agent, by stimulatingthe endogenous miR167 promoter. For example, a transcription factor or achemical agent may be used to elevate gene expression from native orchimeric miR167 promoter, thus inducing the promoter and miR167 geneexpression.

According to the present invention, a desired plant is one that exhibitsimprovement over the control plant (i.e., progenitor, wild type plant)in one or more of the aforementioned parameters. In certain embodiments,the aforementioned parameters are compared between plants grown innitrogen-moderate or nitrogen-rich cultivation conditions. In otherembodiments, the aforementioned parameters are compared between atransgenic plant provided herein and a wild type plant, which are grownin nitrogen conditions in which lateral root growth is repressed in thewild type plant (e.g., nitrogen poor or other nitrogen conditions). Inan embodiment, a desired plant is one that shows at least 5% increaseover the control plant in at least one parameter. In a preferredembodiment, a desired plant is one that shows at least 20% increase overthe control plant in at least one parameter. Most preferred is a plantthat shows at least 50% increase in at least one parameter.

6.12 Cells

Optionally, germ line cells may be used in the methods described hereinrather than, or in addition to, somatic cells. The term “germ linecells” refers to cells in the plant organism which can trace theireventual cell lineage to either the male or female reproductive cell ofthe plant. Other cells, referred to as “somatic cells” are cells whichgive rise to leaves, roots and vascular elements which, althoughimportant to the plant, do not directly give rise to gamete cells.Somatic cells, however, also may be used. With regard to callus andsuspension cells which have somatic embryogenesis, many or most of thecells in the culture have the potential capacity to give rise to anadult plant. If the plant originates from single cells or a small numberof cells from the embryogenic callus or suspension culture, the cells inthe callus and suspension can therefore be referred to as germ cells. Inthe case of immature embryos which are prepared for treatment by themethods described herein, certain cells in the apical meristem region ofthe plant have been shown to produce a cell lineage which eventuallygives rise to the female and male reproductive organs. With many or mostspecies, the apical meristem is generally regarded as giving rise to thelineage that eventually will give rise to the gamete cells. An exampleof a non-gamete cell in an embryo would be the first leaf primordia incorn which is destined to give rise only to the first leaf and none ofthe reproductive structures.

6.13 Plant Regeneration

Following transformation, a plant may be regenerated, e.g., from singlecells, callus tissue or leaf discs, as is standard in the art. Almostany plant can be entirely regenerated from cells, tissues, and organs ofthe plant. Available techniques are reviewed in Vasil et al., 1984, inCell Culture and Somatic Cell Genetics of Plants, Vols. I, II, and III,Laboratory Procedures and Their Applications (Academic Press); andWeissbach et al., 1989, Methods For Plant Mol. Biol.

The transformed plants may then be grown, and either pollinated with thesame transformed strain or different strains, and the resulting hybridhaving expression of the desired phenotypic characteristic identified.Two or more generations may be grown to ensure that expression of thedesired phenotypic characteristic is stably maintained and inherited andthen seeds harvested to ensure expression of the desired phenotypiccharacteristic has been achieved.

Normally, a plant cell is regenerated to obtain a whole plant from thetransformation process. The term “growing” or “regeneration” as usedherein means growing a whole plant from a plant cell, a group of plantcells, a plant part (including seeds), or a plant piece (e.g., from aprotoplast, callus, or tissue part).

Regeneration from protoplasts varies from species to species of plants,but generally a suspension of protoplasts is first made. In certainspecies, embryo formation can then be induced from the protoplastsuspension. The culture media will generally contain various amino acidsand hormones, necessary for growth and regeneration. Examples ofhormones utilized include auxins and cytokinins. Efficient regenerationwill depend on the medium, on the genotype, and on the history of theculture. If these variables are controlled, regeneration isreproducible.

Regeneration also occurs from plant callus, explants, organs or parts.Transformation can be performed in the context of organ or plant partregeneration (see Methods in Enzymology, Vol. 118 and Klee et al.,Annual Review of Plant Physiology, 38:467, 1987). Utilizing the leafdisk-transformation-regeneration method of Horsch et al., Science,227:1229, 1985, disks are cultured on selective media, followed by shootformation in about 2-4 weeks. Shoots that develop are excised from calliand transplanted to appropriate root-inducing selective medium. Rootedplantlets are transplanted to soil as soon as possible after rootsappear. The plantlets can be repotted as required, until reachingmaturity.

In vegetatively propagated crops, the mature transgenic plants arepropagated by utilizing cuttings or tissue culture techniques to producemultiple identical plants. Selection of desirable transgenics is madeand new varieties are obtained and propagated vegetatively forcommercial use.

In seed propagated crops, mature transgenic plants can be self crossedto produce a homozygous inbred plant. The resulting inbred plantproduces seed containing the newly introduced foreign gene(s). Theseseeds can be grown to produce plants that would produce the selectedphenotype, e.g., increased lateral root growth, uptake of nutrients,overall plant growth and/or vegetative or reproductive yields.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences. Transgenic plants expressing the selectable marker can bescreened for transmission of the nucleic acid of the present inventionby, for example, standard immunoblot and DNA detection techniques.Transgenic lines are also typically evaluated on levels of expression ofthe heterologous nucleic acid. Expression at the RNA level can bedetermined initially to identify and quantitate expression-positiveplants. Standard techniques for RNA analysis can be employed and includePCR amplification assays using oligonucleotide primers designed toamplify only the heterologous RNA templates and solution hybridizationassays using heterologous nucleic acid-specific probes. The RNA-positiveplants can then analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium. For transformation and regeneration of maize see,Gordon-Kamm et al., 1990, The Plant Cell, 2:603-618.

Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,1983, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp. 124-176; and Binding,Regeneration of Plants, Plant Protoplasts, 1985, CRC Press, Boca Raton,pp. 21-73.

The regeneration of plants containing the foreign gene introduced byAgrobacterium from leaf explants can be achieved as described by Horschet al., 1985, Science, 227:1229-1231. In this procedure, transformantsare grown in the presence of a selection agent and in a medium thatinduces the regeneration of shoots in the plant species beingtransformed as described by Fraley et al., 1983, Proc. Natl. Acad. Sci.(U.S.A.), 80:4803. This procedure typically produces shoots within twoto four weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, A. Weissbach and H. Weissbach, eds., 1988,Academic Press, Inc., San Diego, Calif. This regeneration and growthprocess includes the steps of selection of transformant cells andshoots, rooting the transformant shoots and growth of the plantlets insoil. For maize cell culture and regeneration see generally, The MaizeHandbook, Freeling and Walbot, Eds., 1994, Springer, New York 1994; Cornand Corn Improvement, 3rd edition, Sprague and Dudley Eds., 1988,American Society of Agronomy, Madison, Wis.

6.14 Plants and Plant Cells

Also provided herein are a plant cell having the nucleotide sequenceconstructs of the invention. A further aspect of the present inventionprovides a method of making such a plant cell involving introduction ofa vector including the construct into a plant cell. For integration ofthe construct into the plant genome, such introduction will be followedby recombination between the vector and the plant cell genome tointroduce the sequence of nucleotides into the genome. RNA encoded bythe introduced nucleic acid construct may then be transcribed in thecell and descendants thereof, including cells in plants regenerated fromtransformed material. A gene stably incorporated into the genome of aplant is passed from generation to generation to descendants of theplant, so such descendants should show the desired phenotype.

In certain embodiments, a plant cell comprises a miR167 nucleotidesequence operably associated with a pericycle specific promoter, whichis optionally a constitutive or inducible promoter. In otherembodiments, a plant cell comprises multiple copies of a miR167 operablyassociated with a pericycle specific promoter. In specific embodimentsprovided herein are plants (and plant cells thereof) that overexpress,constitutionally express and/or inducibly express miR167 in thepericycle of the plant, as compared to other tissues in the plant and/oras compared to a wild type plant.

The present invention also provides a plant comprising a plant cell asdisclosed. Transformed seeds and plant parts are also encompassed.

In addition to a plant, the present invention provides any clone of sucha plant, seed, selfed or hybrid progeny and descendants, and any part ofany of these, such as cuttings, seed. The invention provides any plantpropagule, that is any part which may be used in reproduction orpropagation, sexual or asexual, including cuttings, seed and so on. Alsoencompassed by the invention is a plant which is a sexually or asexuallypropagated off-spring, clone or descendant of such a plant, or any partor propagule of said plant, off-spring, clone or descendant. Plantextracts and derivatives are also provided.

Any species of woody, ornamental or decorative, crop or cereal, fruit orvegetable plant, and algae (e.g., Chlamydomonas reinhardtii) may be usedin the compositions and methods provided herein. Non-limiting examplesof plants include plants from the genus Arabidopsis or the genus Oryza.Other examples include plants from the genuses Acorus, Aegilops, Allium,Amborella, Antirrhinum, Apium, Arachis, Beta, Betula, Brassica,Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia,Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus,Hordeum, Ipomoea, Lactuca, Linum, Liriodendron, Lotus, Lupinus,Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar, Pennisetum,Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus, Populus,Prunus, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum, Solanum,Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria, Triticum, Vitis,Zea, or Zinnia.

Plants included in the invention are any plants amenable totransformation techniques, including gymnosperms and angiosperms, bothmonocotyledons and dicotyledons.

Examples of monocotyledonous angiosperms include, but are not limitedto, asparagus, field and sweet corn, barley, wheat, rice, sorghum,onion, pearl millet, rye and oats and other cereal grains.

Examples of dicotyledonous angiosperms include, but are not limited totomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers,lettuce, peas, alfalfa, clover, cole crops or Brassica oleracea (e.g.,cabbage, broccoli, cauliflower, brussel sprouts), radish, carrot, beets,eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers andvarious ornamentals.

Examples of woody species include poplar, pine, sequoia, cedar, oak,etc.

Still other examples of plants include, but are not limited to, wheat,cauliflower, tomato, tobacco, corn, petunia, trees, etc.

In certain embodiments, plants of the present invention are crop plants(for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice,sorghum, millet, cassaya, barley, pea, and other root, tuber, or seedcrops. Exemplary cereal crops used in the compositions and methods ofthe invention include, but are not limited to, any species of grass, orgrain plant (e.g., barley, corn, oats, rice, wild rice, rye, wheat,millet, sorghum, triticale, etc.), non-grass plants (e.g., buckwheatflax, legumes or soybeans, etc.). Grain plants that provide seeds ofinterest include oil-seed plants and leguminous plants. Other seeds ofinterest include grain seeds, such as corn, wheat, barley, rice,sorghum, rye, etc. Oil seed plants include cotton, soybean, safflower,sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Other importantseed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, andsorghum. Leguminous plants include beans and peas. Beans include guar,locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, limabean, fava bean, lentils, chickpea, etc.

Horticultural plants to which the present invention may be applied mayinclude lettuce, endive, and vegetable brassicas including cabbage,broccoli, and cauliflower, and carnations and geraniums. The presentinvention may also be applied to tobacco, cucurbits, carrot, strawberry,sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus, and pine.

The present invention may be used for transformation of other plantspecies, including, but not limited to, corn (Zea mays), canola(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum, Nicotianabenthamiana), potato (Solanum tuberosum), peanuts (Arachis hypogaea),cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya(Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley,Arabidopsis spp., vegetables, ornamentals, and conifers.

6.15 Cultivation

One skilled in the art knows what constitute nitrogen-poor andnitrogen-rich growth conditions for the cultivation of most, if not all,important crop and ornamental plants. For example, for the cultivationof wheat see Alcoz et al., 1993, Agronomy Journal 85:1198-1203; Rao andDao, 1992, J. Am. Soc. Agronomy 84:1028-1032; Howard and Lessman, 1991,Agronomy Journal 83:208-211; for the cultivation of corn see Tollenearet al., 1993, Agronomy Journal 85:251-255; Straw et al., Tennessee Farmand Home Science: Progress Report, Spring 1993, 166:20-24; Miles, S. R.,1934, J. Am. Soc. Agronomy 26:129-137; Dara et al., 1992, J. Am. Soc.Agronomy 84:1006-1010; Binford et al., 1992, Agronomy Journal 84:53-59;for the cultivation of soybean see Chen et al., 1992, Canadian Journalof Plant Science 72:1049-1056; Wallace et al., 1990, Journal of PlantNutrition 13:1523-1537; for the cultivation of rice see Oritani andYoshida, 1984, Japanese Journal of Crop Science 53:204-212; for thecultivation of linseed see Diepenbrock and Porksen, 1992, IndustrialCrops and Products 1: 165-173; for the cultivation of tomato seeGrubinger et al., 1993, Journal of the American Society forHorticultural Science 118:212-216; Cerne, M., 1990, Acta Horticulture277:179-182; for the cultivation of pineapple see Magistad et al., 1932,J. Am. Soc. Agronomy 24:610-622; Asoegwu, S, N., 1988, FertilizerResearch 15:203-210; Asoegwu, S. N., 1987, Fruits 42:505-509; for thecultivation of lettuce see Richardson and Hardgrave, 1992, Journal ofthe Science of Food and Agriculture 59:345-349; for the cultivation ofmint see Munsi, P. S., 1992, Acta Horticulturae 306:436-443; for thecultivation of camomile see Letchamo, W., 1992, Acta Horticulturae306:375-384; for the cultivation of tobacco see Sisson et al., 1991,Crop Science 31:1615-1620; for the cultivation of potato see Porter andSisson, 1991, American Potato Journal, 68:493-505; for the cultivationof brassica crops see Rahn et al., 1992, Conference “Proceedings, secondcongress of the European Society for Agronomy” Warwick Univ., p.424-425; for the cultivation of banana see Hegde and Srinivas, 1991,Tropical Agriculture 68:331-334; Langenegger and Smith, 1988, Fruits43:639-643; for the cultivation of strawberries see Human and Kotze,1990, Communications in Soil Science and Plant Analysis 21:771-782; forthe cultivation of sorghum see Mahalle and Seth, 1989, Indian Journal ofAgricultural Sciences 59:395-397; for the cultivation of plantain seeAnjorin and Obigbesan, 1985, Conference “International Cooperation forEffective Plantain and Banana Research” Proceedings of the thirdmeeting. Abidjan, Ivory Coast, p. 115-117; for the cultivation of sugarcane see Yadav, R. L., 1986, Fertiliser News 31:17-22; Yadav and Sharma,1983, Indian Journal of Agricultural Sciences 53:38-43; for thecultivation of sugar beet see Draycott et al., 1983, Conference“Symposium Nitrogen and Sugar Beet” International Institute for SugarBeet Research—Brussels Belgium, p. 293-303. See also Goh and Haynes,1986, “Nitrogen and Agronomic Practice” in Mineral Nitrogen in thePlant-Soil System, Academic Press, Inc., Orlando, Fla., p. 379-468;Engelstad, O. P., 1985, Fertilizer Technology and Use, Third Edition,Soil Science Society of America, p. 633; Yadav and Sharmna, 1983, IndianJournal of Agricultural Sciences, 53:3-43.

6.16 Products of Transgenic Plants

Engineered plants exhibiting the desired physiological and/or agronomicchanges can be used directly in agricultural production.

Thus, provided herein are products derived from the transgenic plants ormethods of producing transgenic plants provided herein. In certainembodiments, the products are commercial products. Some non-limitingexample include genetically engineered trees for e.g., the production ofpulp, paper, paper products or lumber; tobacco, e.g., for the productionof cigarettes, cigars, or chewing tobacco; crops, e.g., for theproduction of fruits, vegetables and other food, including grains, e.g.,for the production of wheat, bread, flour, rice, corn; and canola,sunflower, e.g., for the production of oils.

In certain embodiments, commercial products are derived from agenetically engineered (e.g., comprising overexpression of miR167 in thepericycle of the plant) species of woody, ornamental or decorative, cropor cereal, fruit or vegetable plant, and algae (e.g., Chlamydomonasreinhardtii), which may be used in the compositions and methods providedherein. Non-limiting examples of plants include plants from the genusArabidopsis or the genus Oryza. Other examples include plants from thegenuses Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium,Arachis, Beta, Betula, Brassica, Capsicum, Ceratopteris, Citrus,Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus, Glycine,Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca, Linum,Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago, Mesembryanthemum,Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus, Physcomitrella, Picea,Pinus, Poncirus, Populus, Prunus, Robinia, Rosa, Saccharum, Schedonorus,Secale, Sesamum, Solanum, Sorghum, Stevia, Thellungiella, Theobroma,Triphysaria, Triticum, Vitis, Zea, or Zinnia.

In some embodiments, commercial products are derived from a geneticallyengineered (e.g., comprising overexpression of miR167 in the pericycleof the plant) gymnosperms and angiosperms, both monocotyledons anddicotyledons. Examples of monocotyledonous angiosperms include, but arenot limited to, asparagus, field and sweet corn, barley, wheat, rice,sorghum, onion, pearl millet, rye and oats and other cereal grains.Examples of dicotyledonous angiosperms include, but are not limited totomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers,lettuce, peas, alfalfa, clover, cole crops or Brassica oleracea (e.g.,cabbage, broccoli, cauliflower, brussel sprouts), radish, carrot, beets,eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers andvarious ornamentals.

In certain embodiments, commercial products are derived from agenetically engineered (e.g., comprising overexpression of miR167 in thepericycle of the plant) woody species, such as poplar, pine, sequoia,cedar, oak, etc.

In other embodiments, commercial products are derived from a geneticallyengineered (e.g., comprising overexpression of miR167 in the pericycleof the plant) plant including, but are not limited to, wheat,cauliflower, tomato, tobacco, corn, petunia, trees, etc.

In certain embodiments, commercial products are derived from agenetically engineered (e.g., comprising overexpression of miR167 in thepericycle of the plant) crop plants, for example, cereals and pulses,maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassaya, barley,pea, and other root, tuber, or seed crops. In one embodiment, commercialproducts are derived from a genetically engineered (e.g., comprisingoverexpression of miR167 in the pericycle of the plant) cereal crops,including, but are not limited to, any species of grass, or grain plant(e.g., barley, corn, oats, rice, wild rice, rye, wheat, millet, sorghum,triticale, etc.), non-grass plants (e.g., buckwheat flax, legumes orsoybeans, etc.). In another embodiments, commercial products are derivedfrom a genetically engineered (e.g., comprising overexpression of miR167in the pericycle of the plant) grain plants that provide seeds ofinterest, oil-seed plants and leguminous plants. In other embodiments,commercial products are derived from a genetically engineered (e.g.,comprising overexpression of miR167 in the pericycle of the plant) grainseed plants, such as corn, wheat, barley, rice, sorghum, rye, etc. Inyet other embodiments, commercial products are derived from agenetically engineered (e.g., comprising overexpression of miR167 in thepericycle of the plant) oil seed plants, such as cotton, soybean,safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Incertain embodiments, commercial products are derived from a geneticallyengineered (e.g., comprising overexpression of miR167 in the pericycleof the plant) oil-seed rape, sugar beet, maize, sunflower, soybean, orsorghum. In some embodiments, commercial products are derived from agenetically engineered (e.g., comprising overexpression of miR167 in thepericycle of the plant) leguminous plants, such as beans and peas (e.g.,guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean,lima bean, fava bean, lentils, chickpea, etc.)

In certain embodiments, commercial products are derived from agenetically engineered (e.g., comprising overexpression of miR167 in thepericycle of the plant) horticultural plant, such as lettuce, endive,and vegetable brassicas including cabbage, broccoli, and cauliflower,and carnations and geraniums; tomato, tobacco, cucurbits, carrot,strawberry, sunflower, tomato, pepper, chrysanthemum, poplar,eucalyptus, and pine.

In still other embodiments, commercial products are derived from agenetically engineered (e.g., comprising overexpression of miR167 in thepericycle of the plant) corn (Zea mays), canola (Brassica napus,Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum, Nicotiana benthamiana), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), oats, barley, Arabidopsis spp., vegetables,ornamentals, and conifers.

6.16 Kits

In one aspect, the present invention provides any of the above-mentionedcompositions in kits, optionally including instructions for use of thecomposition e.g., for the inhibition of a gene. The “kit” typicallydefines a package including one or more compositions of the inventionand the instructions, and/or analogs, derivatives, or functionallyequivalent compositions thereof. Thus, for example, the kit can includea description of use of the composition for participation in anytechnique associated in the inhibition of genes. The kit can include adescription of use of the compositions as discussed herein. Instructionsalso may be provided for use of the composition in any suitabletechnique as previously described. The instructions may be of any formprovided in connection with the composition.

The kits described herein may also contain one or more containers, whichmay contain the inventive composition and other ingredients aspreviously described. The kits also may contain instructions for mixing,diluting, and/or administrating the compositions in some cases. The kitsalso can include other containers with one or more solvents,surfactants, preservative and/or diluents (e.g., normal saline (0.9%NaCl), or 5% dextrose) as well as containers for mixing, diluting and/oradministrating the compositions.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the composition may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are used, the liquidform may be concentrated or ready to use. The solvent will depend on theactive compound(s) within the composition. Suitable solvents are wellknown, for example as previously described, and are available in theliterature.

The invention also involves, in another aspect, promotion of theinhibition of miR167-regulated genes according to any of the systems ormethods described herein. As used herein, “promoted” includes allmethods of doing business including methods of education, hospital andother clinical instruction, pharmaceutical industry activity includingpharmaceutical sales, and any advertising or other promotional activityincluding written, oral and electronic communication of any form,associated with compositions of the invention. “Instructions” can definea component of promotion, and typically involve written instructions onor associated with packaging of compositions of the invention.Instructions also can include any oral or electronic instructionsprovided in any manner.

7. EXAMPLE Ectopic Overexpression of miRNA167 in Plants Causes anIncrease in Lateral Root Growth

7.1 Summary

By assaying gene expression at the single cell level using acell-specific technique developed by Birnbaum et al., 2003, Science 302,1956-60, that is particularly sensitive for revealing gene regulation,we have been able to determine that thousands of new genes are regulatedat the level of transcription in response to nitrogen treatment.Previous studies on nitrogen-(N—) regulated genes in whole root samplesfailed to uncover these genes. Since the newly discovered N-regulatedgenes included targets of known microRNAs, we postulated that microRNAscould be involved in regulating the levels of target mRNAs in responseto nitrogen treatment, and that the miRNAs themselves could be regulatedby nitrogen. This is indeed true for the case study miR, miR167. Usingthe network modeling tools developed by Gutierrez et al., 2007, GenomeBiol 8, R7, we have also been able, for the first time, to confirm theassociation of a microRNA with a N-regulated gene network. Further, wehave shown that miR167 controls N-regulation of an auxin-responsetranscription factor ARF8, which itself controls a variety ofN-responsive target genes. Most importantly, using overexpressor miR167lines of Arabidopsis and knockout arf8 lines of Arabidopsis, we haveshown that miR167 controls a key switch involved in regulating rootarchitecture in response to nitrogen treatment. It is noted that theArabidopsis model used herein is known in the art to be a model plantsystem for other plant systems.

It had been shown previously that a mutation in ARF8 resulted in plantsthat showed increased emergence of lateral roots (Tian et al., 2004,Plant J 40, 333-43). However, these authors did not examine nutrientregulation of ARF8, nor elaborate their phenotypic characterization toshow that ARF8 acts as a critical checkpoint to regulate the balancebetween lateral root initiation and emergence in response to nitrogentreatment. It had also been shown that miR167 regulates levels of ARF8mRNA during flower development (Wu et al., 2006, Development 133,4211-8). However, the presence of miR167 and the miR167-ARF8relationship was not examined or tested in roots. Thus, the linking ofnitrogen regulation of gene expression to miR167 and its role in thenitrogen regulation of ARF8 in the context of lateral root developmentis entirely new.

Using systems biology/network approaches to analyze the microarray datagenerated from N-treated plant roots, we identified the predicted mRNAtargets of miR167, and developed a model for how nitrogen represseslateral root emergence via miR167 (FIG. 2B). The phenotypic effect ofnitrogen applications on lateral root emergence results from the actionof the miR167 to its targets to enable degradation mRNA for a group of‘checkpoint’ genes that normally down-regulate lateral root emergencewhen nitrogen is replete. These targets include ARF8 (At5g37020) andpotentially other target genes (including ARF6 (At1g30330), At2g48110,At3g19290, At3g42100 and At3g61310). Since miR167 appears to control alarge group of target gene mRNAs, it is a key regulator, which has thepower to control a circuit of genes involved in modulating plant rootdevelopment and potentially also nutrient uptake and metabolic capacity.Targeting just a single factor, miR167, for modification in transgenicplants will thus allow the control a developmentally connected circuitof many genes at once.

7.2 Materials and Methods

Plant material. Arabidopsis Co10 GFP-expressing root cell lines markingthe lateral root cap (E4722), epidermis and cortex (E1001), endodermisand pericycle (E470) and pericycle (E3754) were obtained from theEnhancerTraps collection developed by Dr. Scott Poethig (see worldwideweb at enhancertraps.bio.upenn.edu) through the Arabidopsis BiologicalResource Center (ABRC) at Ohio State University (FIG. 6). AGFP-expressing line marking the stele (pWOL::GFP:stele) was obtainedfrom Bonke et al., 2003, Nature 426, 181-6. gARF8::GUS, MARF::GUS,P_(MIR167a)::GUS, P_(MIR167b)::GUS, arf8-3, arf6-2, a hemizygouspopulation of arf6-2 arf6-2 arf8-3/+, and T1 transformants expressing35S::miR167a were all kindly provided by Dr. Jason Reed¹⁷ (Tian et al.,2004, Plant Physiol 135, 25-38). A GFP-tagged line reporting cytosolicGlutamine Synthetase (At5g37600) expression was obtained from ABRC(stock CS36947).

Plant growth and treatment. All experiments were carried out intriplicate. Approximately 6,000 seeds (per replicate) of each GFP lineused for sorted cell experiments or of Co10 for whole-root andprotoplasting controls were sterilized and sown on Nitex 03-250/47 mesh(Sefar America, Bricarcliff Manor, N.Y., USA). The mesh was supported ona custom-built platform for hydroponic tissue culture inside a Phytatray(Sigma-Aldrich, St. Louis, Mo., USA) containing nitrogen andsucrose-free1× Murashige and Skoog basal medium (custom-produced byGibcoBRL, Gaithersburg, Md., USA) supplemented with 3 mM sucrose and 0.5mM ammonium succinate (1 mM ammonium). All components were kept sterilethroughout the growth period of a 16 hr light (50 mmol photons m⁻²s⁻¹light intensity)/8 hr dark cycles at 22° C. which was maintained insidea growth incubator (Percival Scientific Inc., Perry, Iowa, USA).Approximately 200 seeds of each ARF8- and miR167-related line, and Co10in ARF8/miR167 experiments were sown in a similar fashion, but with 0.2mM ammonium succinate. For treatments, 12 days after plants were placedinto a growth chamber seedlings were treated by adding KNO₃ to a finalconcentration of 5 mM; control plants were mock-treated by adding thesame concentration of KCl. For MSX-experiments plants were additionallytreated with 5 mM glutamate, 5 mM glutamine, and/or 1 mM methylsulfoximine (MSX) following Barabasi et al., 2004, Network biology:understanding the cell's functional organization. Nat Rev Genet. 5,101-13. For isolation of GFP-expressing cells and mock-sortedprotoplasts, seedling roots were harvested and subject to enzymaticdigestion (see below, as Birnbaum et al., 2005, Nat Methods 2, 615-9).Otherwise whole roots were harvested and frozen immediately in N₂(1)prior to RNA extraction, or harvested and incubated for 60 minutes in asolution identical to that used for protoplasting (as Birnbaum et al.,2005, Nat Methods 2, 615-9) with the exception of pectolyase andcellulysin enzymes, then frozen in N₂(1). For microarray analysis ofarf6-2/arf8-3 and 35S::miR167a, 100 seeds of the segregatingarf6-2/arf6-2 arf8-3/+ seed line and 300 seeds of the T1 35S::miR167aseed line were sown, seedlings treated as above with KNO₃ or KCl, thenroots and shoots immediately harvested and frozen separately for eachindividual seedling. DNA from each shoot sample was extracted using theQiagen DNeasy Plant Mini Kit isolation kit (Qiagen, Hilden, Germany)according to manufacturers instructions. arf6-2/arf6-2 arf8-3/+DNAsamples were PCR genotyped to identify seedlings that carried two copiesof the arf8-3 allele using primers previously described in Wu et al.,2006, Development 133, 4211-8. T1 35S::miR167a DNA samples were PCRgenotyped to detect the presence of the 35S::miR167a transgene usingoligos designed to detect the BAR gene (5′-TCAGTTCCAAACGTAAAACGG-3′ (SEQID NO:1) and 5′-CGTACCGAGCCGCAGGAAC-3′ (SEQ ID NO:2)). RNA from seedlingroots genotyped to be positive in each case was then extracted (seebelow).

Histology and microscopy. Treated and control-treated 12 day oldseedlings were removed from the mesh and X-gluc activity assayed foraccording to Sessions et al., 1999, Plant J 20, 259-63. GUS-stainedseedling roots were mounted in dH₂O and viewed using Zeiss Axioskopmicroscope (Zeiss, Jena, Germany). Images were taken with a colordigital Zeiss Axiocam camera using the Zeiss Axiovision software.GFP-expressing seedlings were viewed using a Leica TCS SP2 LaserScanning Spectral Confocal Microscope system (Leica, Leica MicrosystemsGmbH, Germany). Adobe Photoshop was used to crop digital images. Forassay of lateral root outgrowth 24 Co10 and 12 arf8 seedlings fromphytatrays that had been supplemented at the start of the light periodon day 12 with 5 mM KNO₃ or not supplemented (control) were removed onday 16 (4 days after treatment), mounted in dH₂O, and visualized using aNikon Eclipse 90i microscope (Nikon, Tokyo, Japan). The number of (i)stage I to IV lateral root primordia, (ii) stage V to VII lateral rootprimordia, (iii) emerging lateral root primordia and (iv) fully emergedlateral roots (all according to Malamy et al, 1997, Development 124,33-44) on each root were scored. During analysis a comparison was madebetween the total numbers of initiating lateral root primordia (i andii) and emerging/emerged (iii and iv) lateral roots. For analysis of the35S::miR167a-segregating line treatment was carried out as above, thenon the 18^(th) day approximately 160 seedlings were individuallyharvested, the number of lateral roots counted for each while the leaveswere frozen in liquid in N₂ (1). DNA was extracted from each leaf sampleand genotyped for the presence of the 35S::miR167a-transgene in order toidentify 35S::miR167a seedlings.

Plant cell protoplasting and Fluorescence Activated Cell Sorting.Immediately following the two hour treatment period roots were harvestedand protoplasted according to established techniques (Birnbaum et al.,2005, Nat Methods 2, 615-9). During this time the KNO₃-treatment ofroots and resulting cells from KNO₃-treated seedlings was eithercontinued (continuous treatment), or discontinued (transitory treatment)whilst protoplasting and subsequent cell-sorting was carried out. ForMSX treatments of pericycle cells the KNO₃/MSX treatment or the(KCl)/MSX-control treatment was either continued (continuous-MSX andCC-continuous-MSX), or not (transitory-MSX and CC-transitory-MSX).GFP-expressing cells were isolated on a Cytomation MoFlo fluorescenceactivated cell sorter (Cytomation, Fort Collins, Colo., USA) directlyinto lysis buffer, mixed and immediately frozen at −80° C. for RNAextraction according to Birnbaum et al., 2005, Nat Methods 2, 615-9; thenon GFP-expressing sorted cells from each sort were also collected. Inparallel, Co10 whole root protoplasts were isolated and stored on icefor one hour to mimic the cell sorting procedure. During this time equalvolumes of sample were removed into lysis buffer and immediately frozenat −80° C. at 20 min intervals; for RNA extraction the three sampleswere pooled.

RNA isolation, quantitative PCR and microarray analysis. RNA extractionfrom sorted or protoplasted cells, as well as from small amounts of roottissue (from the ARF8/miR167 experiments) was carried out using theQiagen RNAeasy RNA cleanup kit according to manufacturer's instructions.RNA from large amounts of whole roots was extracted with TRIzol(Invitrogen, Carlsbad, Calif.) according to manufacturer's instructions.Standard Affymetrix protocols were then used for amplifying, labelingand hybridizing RNA samples to the ATH1 GeneChip (Affymetrix, SantaClara, Calif., USA). 50 ng RNA from sorted cells and arf6-2/arf8-3 or35S::miR167a whole roots, or 1 μg RNA from protoplasted cells and allother whole root samples was used for hybridisation; the Affymetrixsmall sample labeling protocol was used to amplify the 50 ng RNAsamples. For quantitative RT-PCR confirmation a separate aliquot of thesame sample was assayed. Double stranded cDNA was synthesized using theInvitrogen Thermoscript RT-PCR system according to manufacturer'sinstructions; for 50 ng RNA samples the RNA was first amplified usingthe Affymetrix small sample labeling protocol, and random primersinstead of oligo dT were used for cDNA synthesis priming. QuantitativeRT-PCR was carried out using DNA Master SYBR green labeling on a RocheLightCycler (Roche Applied Science, Mannheim, Germany) according tomanufacturer's instructions. The mRNA concentration for each assayedgene was determined by normalizing expression levels relative to thehighly expressed house-keeping gene Clathrin (At4g24550) and determiningthe quantity of mRNA according to a standard curve for each primer pair.Expression of 12 transcripts including ARF8 was tested for confirmationof genome chip results (not shown). Quantification of miR167a andmiR160a expression was determined using oligos designed against themiR167a precursor (miR167aF: 5′-TCAGATGCCGGTGCACCATA-3′ (SEQ ID NO:3)and miR167aR: 5′-CACCAAGTTTCGAGTAGACCGTGA-3′ (SEQ ID NO:4) (as used inWu et al., 2006, Development 133, 4211-8), and the miR160a precursor(mi160aF: 5′-GTATGCCTGGCTCCCTG-3′ (SEQ ID NO:5) and miR160a R,5′-TCGATGACCTCCGTGG-3′ (SEQ ID NO:6)). Quantification of mARF::GUSexpression was determined by quantifying expression of the GUS gene.Clathrin and GUS were assayed using primers and probes designed by andobtained from TIB Molbiol LLC (Adelphia, N.J.), using the Roche DNAMaster Hybridisation Probes kit.

Microarray Expression Normalization. Transcript expression wasnormalized using the freely available dChip software (see worldwide webat dchip.org). The reproducibility of replicates was analyzed using thecorrelation coefficient and r² value of replicates pairs in the S-PLUS7.0 software package (Insightful Corp., Seattle, Wash., USA). Fordetermination of gene presence or absence a log 2 signal value cutoff of6 was determined by examining the signal values of 25 genes that exhibitwell-characterised cell-specific expression patterns across the fivecell populations examined (FIG. 5).

ANOVA analysis and determination of significance in cell populations.All 22,746 genes that are represented on the ATH1 GeneChip (Affymetrix,Santa Clara, Calif.) were subjected to an ANOVA analysis in order tofind the genes that showed the highest probability of responding totreatments within cell populations or across cell populations. Thefollowing statistical analysis was implemented in MATLAB (The MathWorks,Natick, Mass., USA). (i) ANOVA filtering step. We first filtered data toobtain a list of genes that showed the most consistently variablesignals with respect to treatments. We modeled a two way ANOVA with thecells as the first factor and treatments as the second factor(Y=μ+α_(cell pop)+α_(cell pop)+α_(cell pop*treatment)+ε, where Y is theexpression of a gene represented by the normalized dChip signal, μ isthe global mean and the alpha coefficients correspond to the effects ofcell population, treatment and the interaction between cell populationand treatment). We then used ANOVA test statistics for both treatment ortreatment x cell population interactions to determine the genes with a pvalue ≦0.05. Next we removed genes which had an ambiguous match toAffymetrix probe sequences according to the latest annotation fileavailable from Affymetrix. We also removed any genes found to beaffected by the protoplasting treatment in previous work (Birnbaum etal., 2003, Science 302, 1956-60. (ii) We then subjected the genesshowing a treatment x cell population interaction to FDR analysis asimplemented by Significance of Analysis of Microarrays (Tusher et. al,2001, Proc Natl Acad Sci USA 98, 5116-21). Each category was testedseparately (five cell population categories for cell x treatmentresponsive). The Wilcoxon test statistic was used with the maximumnumber of possible iterations (720). Only genes that showed a falsediscovery rate of 5% or less in any category were kept on the list ofnitrogen-responsive genes. This enabled us to generate a categorizationfor each gene showing the cell population(s) that it was N-responsive inand the direction of N-response (induction or repression). For genesshowing a treatment effect only we used the difference between controland treat samples to determine whether this effect was an N-induction orrepression. We followed a similar procedure for both the whole roottreated and the root protoplasted treated datasets. The two independentexperiments revealed almost exactly the same clusters corroborating theresponse clusters found by the statistical analysis. We used Euclideanclustering to group N-response clusters. For statistical analysis of theeffect of miR167a overexpression, ARF8 or ARF6/8 knockout, or MSXtreatment, clusters of genes of interest were analyzed by carrying outan un-paired t-test using the Wilcoxon test statistic with the maximumnumber of possible iterations. We also used chi-squared tests to examinethe proportions of initiating vs. emerging lateral roots.

Network Analysis. We used the VirtualPlant online software (seeworldwide web at virtualplant.org) to carry out analysis of gene listsand for network analysis of our N-regulated genes. The multinetwork thatwe queried to generate N-regulated networks contains information aboutthe way that genes/proteins/metabolites are connected via metabolic,transport, protein:protein, miR:RNA and DNA-protein (regulatory) edges(described in Little et al, 2005, Proc Natl Acad Sci USA 102, 13693-8).The edges are drawn based on information in number of databases, datapublished in the literature, and additional predictions forprotein:protein and miR:RNA interactions. In addition the latest versionof the multinetwork on VirtualPlant contains DNA-protein interactionsbased on the presence of at least one cis element in the promoter of thetarget gene, combined with co-regulation of the target and regulatorgene across all Arabidopsis microarray data that is available in theNASC repository.

7.3 Results and Discussion

Nitrate is a key required nutrient for the synthesis of amino acids,nucleotides and vitamins and is commonly considered to be the mostlimiting for normal plant growth (Vitousek et al., 1991, Biogeochemistry13:87-115). Nitrogenous fertilizer is usually supplied as ammoniumnitrate, potassium nitrate, or urea. Plants are keenly sensitive tonitrogen levels in the soil and, atypically of animal development, adopttheir body plan to cope with their environment (Lopez-Bucio et al.,2003, Curr Opin Plant Biol 6, 280-7); Malamy et al., 2005, Plant CellEnviron 28, 67-77); Walch-Liu et al., 2006, Ann Bot (Lond) 97, 875-81).For example, mutants in several general nitrogen (N)-assimilation genesaffect root architecture (Little et al., 2005, Proc Natl Acad Sci USA102, 13693-8; Remans et al., 2006, Proc Natl Acad Sci USA 103,19206-11). Transduction of this nitrogen signal is linked to a massiveand concerted gene expression response in the root (Gutierrez et al.,2007, Genome Biol 8, R7; Wang et al., 2003, Plant Physiol 132, 556-67.

Thus, we hypothesized that N depletion followed by a short period of Ninflux (a likely environmental scenario) could elicit highly specificreactions in the plant as a developmental response mediating bothmorphological and metabolic changes in specialized cell types. Recentprogress in cell-specific profiling now allows us to ask how the plantregulates development at the cell specific level, using enzymaticdigestion of cell specific expressing fluorescent lines followed byFACS-cell sorting (Birnbaum et al., 2003, Science 302, 1956-60; Birnbaumet al, 2005, Nat Methods 2, 615-9).

To understand the cell-specific reaction to nitrogen on a global scale,we FACS sorted five specific cell populations immediately following atwo hour transitory N-treatment. We used five GFP-expressing lines thatsample the main cell populations of the root, the lateral root cap,epidermis and cortex, endodermis and pericycle, pericycle alone, and thestele (vascular tissues plus pericycle) (FIG. 7). We grew seedlings onlow levels of ammonium for a period of 12 days to ensure nitrogendepletion at the end time point, then applied a two hour 5 mM nitratetransitory treatment to elicit N-regulation of gene expression (asGutierrez et al., 2007, Genome Biol 8, R7). KCl-treated seedlings wereused as a non-treated control. In order to address information about thepersistence of the N-response, the nitrate treatment was eithercontinued during protoplasting and cell sorting (continuous N) ornitrate was only present prior to cell sorting (transitory N). Inparallel non-protoplasted (whole) Co10 roots were processed in a similarfashion (sustained and transitory N). Microarray data was normalizedusing dChip, filtered to remove low signal value genes, then a two-wayANOVA performed to identify genes that showed the highest probability ofresponding to the N-treatment within cell populations or across cellpopulations at a p value of ≦0.05 (see Materials and Methods). At testat a p value of ≦0.05 was used to determine N-regulation in whole roots.Previous work found protoplasting and cell sorting to have little effecton global levels of gene expression; following this study genes known tobe affected by protoplasting (according to Birnbaum et al., 2003,Science 302, 1956-60) were removed from our analysis.

We found that cell specific profiling has the sensitivity to uncoverN-regulation for thousands of new genes beyond what was previouslyknown, and accurately captures cell-specific reactions in amulticellular organism. We found 5,733 genes only to be N-regulated insorted cells, 1,780 genes to be regulated only in whole roots, and 699genes to be regulated in both roots and cells. The 1,780 genes only inwhole roots are likely to be genes that are strongly N-regulated in acell type that we did not sample. In order to validate our cell-specificdata we took three approaches. Firstly we examined the expression of aset of known cell-specific genes and found them to be expressed in thecorrect cell types (FIG. 5). Secondly we assayed the expression of arandom sample of 12 cells-only N-regulated genes in whole roots by qPCR.We reasoned that if cell-specific, the N-regulation of genes would bebarely visible in whole roots, thus a more sensitive expressiontechnique (qPCR) would be required for detection. All 12 genes werefound to be N-regulated in whole roots as predicted (e.g., 3 shown inFIG. 6); we also confirmed the cell specificity of the N-response ofthree of these genes by assaying their expression in sorted cells versussorted non-GFP-marked cells (not shown). Finally we employed reporterconstructs to confirm predicted patterns of N-regulation. We observedwidespread N-induction for cytosolic glutamine synthetase (data notshown), and pericycle and lateral root induction for ARF8 (FIGS. 3A,D).

We found that continuous- and transitory N-treatments elicited similareffects in sorted cells. This was evident on a global scale by the factthat continuous and transitory N-treatment experiment replicatesclustered together (not shown). We found 3,532 genes to be N-regulatedby both sustained and transitory treatment. 1,333 N-regulated genes wereonly N-regulated in continuous N-treated cells and 2,823 onlyN-regulated in transitory N-treated cells. At the gene level,N-regulation was found to be similar for both treatments. Where thisdiffered regulation appeared to be dampened in the temporary N-treatedcells (not shown), suggesting that the response of genes to nitrate israpid and reversible. Because of this we chose to dissect thesustained-treatment data.

In total we found 6,355 transcripts to be regulated at the cell-specificlevel in a combinatorial fashion (data not shown). To classify responseswe first separated genes that exhibit an overall N-regulated effectacross all cell populations studied (769 genes out of 6,355), from thosethat are N-regulated only in 1-4 cell populations (5,586 genes out of6,355) using a two-way ANOVA. Thus we found that a large number (88%) ofgenes exhibit some degree of cell-specificity in their N-response. Weused a Wilcoxon t test at an FDR rate of ≦5% in order to determine inwhich cell population(s) these cell-specific genes were N-induced ordepressed, then Euclidean clustering to create N-response clusters; wewere able to categories 97% (5,426 of 5,586) of the genes. FIG. 1B showsan overview of the N-regulation pattern of all response clusters thatcontain more than 10 genes. We found a striking range of cell specificN-response clusters. Cluster 1 which is N-induced in all cellpopulations contains the majority of genes that have previously beenfound to be N-regulated (according to Gutierrez et al., 2007, GenomeBiol 8, R7 and Wang et al., 2003, Plant Physiol 132, 556-67). Thisincludes core enzymes involved in reducing nitrate and forming aminoacids: nitrate reductases NR1, NR2, nitrite reductase NiR,NADH-dependent glutamate synthase and asparagine synthetase ASN2.Strikingly, this cluster alone accounts for 46% of all genes within thisdataset that are known to be specifically regulated by nitrate, asopposed to downstream N-metabolites (according to Wang et al., 2004,Plant Physiol 136, 2512-22). This cluster of genes is expressed at ahigh level and induced to a strong degree (FIG. 1Ci). Together thishelps account for the fact that these genes had previously been detectedto be N-regulated. In contrast, the majority of N-regulated nitratetransporters were found to be consistently N-depressed (cluster 16) orinduced in the pericycle and repressed in the stele (cluster 17).N-regulated amino acid transporters were found to be regulated in acell-specific manner, often in more inner cells, and in many differenttypes of patterns across the root (clusters 1, 13, 15, 17). Carbon andnitrogen signaling are closely linked (Palenchar et al., 2004, GenomeBiol 5, R91). Core elements of carbohydrate metabolism and thepentose-phosphate pathway are also N-induced in all cells, while sucrosetransporters are regulated in specific cell populations. Together thissuggests that the root instigates a rapid widespread nitrate-regulationof core enzyme-encoding genes in order to assimilate nitrate and toco-ordinate C/N metabolism. The metabolic products of this response(nitrate, assimilated nitrate and sucrose) could then be selectivelychanneled around the root by cell-specific regulation of theirtransporters. Downstream N-metabolites (amino acids) could then act toregulate developmental programs within particular root types. This isevidenced by the fact that addition of the glutamate-analog MSX whichblocks the enzymatic production of the amino acids glutamate andglutamine (as Rawat et al., 1999, Plant J 19, 143-52) appears to reducethe N-responsiveness of several clusters (FIG. 1B); this effect isalleviated by adding-back Glu or Gln. Regulation of downstreammetabolic/developmental programs appears to include those in the GOcategory ‘photosynthesis’ which was found to be over-represented (pvalue <1^(e-6)) in genes that are N-depressed in the epidermis/cortex(clusters 5, 14) (determined using the ‘BioMaps’ tool described inGutierrez et al., 2007, Genome Biol 8, R7). Plastid genes which areannotated to this category have been found in legumes to be associatedwith a switch from nitrogen source-sink status in the root uponinitiation of symbiosis with N₂-fixing bacteria, and it is an intriguingpossibility that this could occur in Arabidopsis in response to nitrogen(Palma et al., 2006, J Exp Bot 57, 1747-58).

By comparing the levels of gene expression before or after N-treatmentwe found that the type of N-regulation falls into two categories: (i)simple induction/repression where the basal level of gene expression issimilar across all cell populations, then N-induced or repressed in aparticular pattern (e.g., cluster 1, FIG. 1Cii); or (ii) a relativealleviation of induction or repression where the basal level of geneexpression is cell specific, and the ‘after-N’-response is similaracross all cell populations (e.g., cluster 5, FIG. 1E).

We further examined the expression of genes before and after N-treatmentby using Pearson clustering to group genes based on their expression andfound that expression in the endodermis/pericycle cell population wasvery similar to that in the pericycle-alone cell population. Howevergene expression between the two is markedly distinct after N-treatment.A strong induction response in the pericycle is particularly evident(FIG. 1A). Among genes that are induced in the pericycle we found the GOterms ‘cell wall modification during multidimensional cell growth’ and‘transmembrane receptor protein tyrosine kinase signaling pathway’ to beoverrepresented (p<1^(e-3)). Since lateral root development whichinvolves regulation of cell growth is stimulated in the pericycle bynitrate treatment (FIG. 3L) we investigated the possibility thatpericycle N-induced response clusters might regulate this process bytaking a network approach and constructing a network ofpericycle-induced genes.

To validate the cell specific approach we carried out aproof-of-principle study to elaborate our predictions concerningregulation of lateral root development by nitrogen at the cell-specificlevel. Within the pericycle-N-induced network (FIG. 2A) we found asubnetwork containing AUXIN RESPONSE FACTOR 8 (ARF8), a known modulatorof root development (Tian et al., 2004, Plant J 40, 333-43) (FIG. 2B).This suggests that the ARF8 effect on lateral roots, which appears toact as an repressor of LR outgrowth, is N-dependent. ARF8 is a knowntarget of the microRNA miR167 (Wu et al., 2006, Development 133,4211-8), implicating miR167 repression in the N-dependent regulation oflateral root development that occurs in the pericycle cell layer.N-regulation of a microRNAs could represent a new layer of regulatorycontrol for development. We tested this hypothesis by usingGUS-expressing marker lines and transgenic lines obtained from Wu etal., 2006, Development 133, 4211-8, and by assaying expression levelsusing qPCR in whole roots and sorted pericycle cells (data not shown).Firstly we confirmed that ARF8 is N-induced in the pericycle (FIGS. 3A,B, C). We then confirmed that miR167 is both expressed in the pericycle,and N-regulated there by quantifying the expression of the miR167aprecursor by qPCR (FIGS. 3D, E, F); to act as a control we confirmedthat the miR160 precursor, which was found to have no predicted targetswithin our N-regulated gene dataset at a free energy of 0.72 (accordingto Dezulian et al., 2006, Bioinformatics 22, 359-60) was not N-regulated(not shown). We established that miR167 is involved in the N-inductionof ARF8 expression levels since the N-induction of ARF8::GUS expressionwas lost when the miR167 target sequence in ARF8 was mutated(mARF8::GUS) (FIGS. 3G, H, I). Strikingly since the miR and target geneare expressed in the same cells this is an example of modulation of geneexpression rather than complete repression/induction. We found that thisN-responsive network was involved in regulating lateral root developmentby showing that overexpression of miR167a, and loss-of ARF8 function inthe arf8-3 mutant led to N-dependent defects in lateral root development(FIG. 3L). In Co10, N-treatment results in a stimulation of lateral rootinitiation but also a repression of lateral root emergence (FIG. 3L).This suggests that Arabidopsis initiates lateral root primordia underconditions of high nitrate availability, but maintains these primordiain an un-emerged state until conditions of low nitrate require that theyemerge to explore the surroundings in search of nitrogen. To examine aconnection between ARF8 and lateral root development we first determinedthe targets of ARF8 transcriptional regulation (FIG. 2B, FIG. 5). Wewere able to confirm that the majority of our ARF8-predicted targets arelikely real since they are mis-regulated during N-treatment in thearf8-3 background (FIG. 2B, evidence b). arf8-3 roots have enhancedrates of lateral root emergence vs. initiation upon N-treatment (FIG.3L); this increased level of lateral root emergence accounts for the arfphenotype described in Tian et al., 2004, Plant J 40, 333-43. Thus theN-responsiveness of root architecture seems to be less in arf8 comparedto wild-type plants. Therefore ARF8 also appears to act as a checkpointto inhibit lateral root emergence when nitrate is replete. As predicted,35S::miR167a seedlings exhibit a similar phenotype, although theinhibitory effect of N is completely lost. 35S::miR167a seedlings alsohave even fewer lateral roots in total (FIG. 3L, right side). Thissuggests that miR167 acts through other targets aside from ARF8 alone tomodulate lateral root development, some of these targets being inducersof lateral root development. All ARF8 predicted targets were found to bemis-regulated in the arf6-2/8-3 (FIG. 2B, evidence c) and 35S::miR167abackgrounds (FIG. 2B, evidence d), which could help to explain thereduction in lateral root numbers. ARF8 is known to act with anotherauxin response factor, ARF6 (FIG. 2B). While ARF6 was not to be found tobe significantly N-regulated in our studies it is also a predictedmiR167 target (Dezulian et al., 2006, Bioinformatics 22, 359-60) andcould modulate the effects of the N-induced ARF8. In addition miR167could act through another of its predicted targets, At1g61310 (Dezulianet al., 2006, Bioinformatics 22, 359-60) to regulate lateral rootnumbers according to N (FIG. 2B). Finally we found that MSX blocked theN-induction of the majority of this network in nitrate-treatedpericycle-sorted cells, indicating the network to be Glu/Gln-responsive(FIG. 2B). Thus miR167/ARF8 could be the link between Glu signaling,auxin signaling and lateral root development proposed in Walch-Liu etal., 2006, Plant Cell Physiol 47, 1045-57. This also fits with ourhypothesis that the assimilated products of nitrate act as cell-specificregulators to influence cell-specific developmental programs. Thisnitrogen-dependent lateral root response appears to be distinct fromprevious pathways since neither NAC1 (Guo et al., 2005, Plant Cell 17,1376-86), ANR1 (Zhang et al., 1998, Science 279, 407-9), nor ARF7/ARF19(Okushima et al., 2007, Plant Cell 19, 118-30) were found to beregulated by nitrogen in our studies. This network is not enriched forgenes involved in auxin signaling, nor in previously characterised geneswhich affect lateral root development. Thus it does not appear that ARF8is involved directly in the initiation or emergence of lateral roots,but instead in the consequences of controlling root architecture and thedevelopmental state of lateral root primordia. The network does containCyclin A2; 3 which has been shown to be expressed in the root meristem(Imai et al., 2006, Plant Cell 18, 382-96). Overexpression of this genehas been found to retard the mitotic cell cycle in proliferatingtissues, and affect cell expansion to result in root dwarfism (Imai etal., 2006, Plant Cell 18, 382-96). In addition CEL1, a glucanase whichappears to be involved in cellulose biosynthesis, is induced by auxinand expressed in young tissues during cell expansion (Shani et al.,2006, Plant Cell Rep 25, 1067-74). These two genes could thus beinvolved in inducing lateral root initiation according to N, while ARF8itself acts as a checkpoint. These findings are consistent with a growthcheckpoint effect which acts upstream of auxin events that areconstantly signaling to affect positioning and initiation of lateralroot primordia (De Smet et al., 2007, Development 134, 681-90). Thecheckpoint effects of ARF8 in the root could also occur in other organssuch as flowers. arf6/8 mutant flowers as well as 35S::miR167a flowersare sterile (Wu et al., 2006, Development 133, 4211-8). In additionCYC2A; 3 has been found to be expressed in inflorescences (Imai et al.,2006, Plant Cell 18, 382-96). Distinguishing possible checkpoint effectsof ARF8 in flowers and roots, and separating the dual effects of miR167on root growth vs. architecture will be our next step.

To explore factors downstream of the miR167-ARF8 circuit in thepericycle, we tested whether potential ARF8 targets exhibit coordinatedresponses within the pericycle. To build such a list of potentialtargets, we searched for genes that were induced in the pericycle (whereARF8 induction is most dramatic), that had an ARF binding site and thatalso showed moderate correlation (R=0.5) with ARF8 over around 1,900microarray experiments deposited in the NASC database (Craigon et al.,2004, Nucleic Acids Res 32:D575-577). The procedure identified 126potential targets, which are listed in Table 1, below.

TABLE 1 AGI ID Gene description At1g03170 Expressed protein At1g03780Targeting protein-related At1g07970 Expressed protein At1g10640Polygalacturonase At1g11730 Galactosyltransferase family proteinAt1g12570 Glucose-methanol-choline (GMC) oxidoreductase family proteinAt1g14350 Encodes a putative MYB transcription factor involved instomata development At1g15570 Cyclin At1g17110 Ubiquitin-specificprotease 15 (UBP15) gene At1g22180 SEC 14 cytosolic factor familyprotein/phosphoglyceride transfer family protein At1g24260 Member of theMADs box transcription factor family At1g25510 Aspartyl protease familyprotein At1g26330 Expressed protein At1g27370 Similar to squamosapromoter-binding protein-like 11 (SPL11) At1g27360 At1g30490 DominantPHV mutations cause transformation of abaxial leaf fates into adaxialleaf fates At1g32930 Galactosyltransferase family protein At1g35780Expressed protein At1g48100 Glycoside hydrolase family 28protein/polygalacturonase (pectinase) family protein At1g49430 Encodes along chain acyl-CoA synthetase At1g51790 Leucine-rich repeat proteinkinase At1g52200 Expressed protein At1g55690 SEC14 cytosolic factorfamily protein/phosphoglyceride transfer family protein At1g62360 ClassI knotted-like homeodomain protein required for shoot apical meristem(SAM) formation At1g63470 DNA-binding family protein At1g65370 Meprinand TRAF homology domain-containing protein/MATH domain- containingprotein At1g67320 DNA primase At1g70710 Endo-1 At1g72250 Kinesin motorprotein-related At1g73930 Similar to FLJ00229 protein [Homo sapiens](GB:BAB84982 At1g74420 Member of Glycosyltransferase Family- 37At1g75240 Zinc finger homeobox family protein/ZF-HD homeobox familyprotein At1g76420 Identified in an enhancer trap line At1g77110 Auxintransport protein (PIN6) mRNA At1g77720 Protein kinase family proteinAt1g79350 DNA-binding protein At1g79420 Expressed protein At2g01210Leucine-rich repeat transmembrane protein kinase At2g02540 Zinc fingerhomeobox family protein/ZF-HD homeobox family protein At2g07170 Similarto expressed protein [Arabidopsis thaliana] (TAIR:At4g27060 At2g07690Minichromosome maintenance family protein/MCM family protein At2g16250Leucine-rich repeat transmembrane protein kinase At2g17930 FATdomain-containing protein/phosphatidylinositol 3- and 4-kinase familyprotein At2g20100 Ethylene-responsive family protein At2g20300 Proteinkinase family protein At2g21050 Amino acid permease At2g23700 Expressedprotein At2g25060 Plastocyanin-like domain-containing protein At2g26180Calmodulin-binding family protein At2g26330 Homologous to receptorprotein kinases At2g27040 PAZ domain-containing protein/piwidomain-containing protein At2g27980 Expressed protein At2g31320 Poly(ADP-ribose) polymerase At2g32590 Barren family protein At2g33560Spindle checkpoint protein-related At2g34710 Dominant PHB mutationscause transformation of abaxial leaf fates into adaxial leaf fatesAt2g35340 RNA helicase At2g36200 Kinesin motor protein-related At2g38160Expressed protein At2g42120 DNA polymerase delta small subunit-relatedAt2g44440 Emsy N terminus domain-containing protein/ENTdomain-containing protein At2g44830 Protein kinase At2g45870 Expressedprotein At3g02110 Serine carboxypeptidase S10 family protein At3g02210Phytochelatin synthetase family protein/COBRA cell expansion proteinCOBL3 At3g02640 Expressed protein At3g05750 Similar to expressed protein[Arabidopsis thaliana] (TAIR:At5g26910 At3g06130 Heavy-metal-associateddomain-containing protein At3g06220 Transcriptional factor B3 familyprotein At3g10310 Kinesin motor protein-related At3g11000 Expressedprotein At3g13000 Expressed protein At3g13510 Expressed proteinAt3g14980 PHD finger transcription factor At3g15550 Expressed proteinAt3g16170 Acyl-activating enzyme 13 (AAE13) At3g17840 Arabidopsisthaliana AT3g17840/MEB5_6 mRNA At3g20070 Encodes a plant-specificprotein of unknown function At3g21310 Expressed protein At3g26932Similar to double-stranded RNA-binding domain (DsRBD)-containing proteinAt5g41070 At3g29280 Expressed protein At3g32400 Formin homology 2domain-containing protein/FH2 domain-containing protein At3g45610Dof-type zinc finger domain-containing protein At3g50890 Zinc fingerhomeobox family protein/ZF-HD homeobox family protein At3g57670 Zincfinger (C2H2 type) protein (WIP2) At3g57830 Leucine-rich repeattransmembrane protein kinase At3g57920 Squamosa promoter-binding proteinAt3g61310 DNA-binding family protein At4g02800 Expressed proteinAt4g11450 Expressed protein At4g13710 Pectate lyase family proteinAt4g14330 Phragmoplast-associated kinesin-related protein 2 (PAKRP2)At4g17000 Expressed protein At4g18020 Pseudo-response regulator 2(APRR2) (TOC2) At4g18820 Expressed protein At4g21326 Subtilase familyprotein At4g21430 Similar to transcription factor jumonji (jmjC)domain-containing protein At1g62310 At4g21550 Transcriptional factor B3family protein At4g25110 Similar to latex-abundant family protein(AMC1)/caspase family protein At1g02170 At4g29030 Glycine-rich proteinAt4g30130 Expressed protein At4g32730 Encodes a putative c-myb-liketranscription factor with three MYB repeats At4g37750 Ovule developmentprotein aintegumenta (ANT) At4g39010 Glycosyl hydrolase family 9 proteinAt5g02370 Kinesin motor protein-related At5g03680 Trihelix DNA-bindingprotein At5g07180 Arabidopsis receptor-like kinase At5g07800Flavin-containing monooxygenase family protein/FMO family proteinAt5g08390 Transducin family protein/WD-40 repeat family proteinAt5g11160 Adenine phosphoribosyltransferase At5g11510 AF371975Arabidopsis thaliana putative c-myb-like transcription factor MYB3R-4At5g20540 Expressed protein At5g20740 Invertase/pectin methylesteraseinhibitor family protein At5g25090 Plastocyanin-like domain-containingprotein At5g26850 Similar to cyclin-related [Arabidopsis thaliana](TAIR:At2g41830 At5g27680 DNA helicase At5g33370 GDSL-motiflipase/hydrolase family protein At5g35930 AMP-dependent synthetase andligase family protein At5g37020 Auxin-responsive factor (ARF8) At5g43080Cyclin At5g51560 Leucine-rich repeat transmembrane protein kinaseAt5g52860 ABC transporter family protein At5g56740 Histoneacetyltransferase family protein At5g60210 Cytoplasmic linkerprotein-related At5g60910 Agamous-like MADS box protein AGL8/FRUITFULL(AGL8) At5g64980 Expressed protein At5g67110 Basic helix-loop-helix(bHLH) family protein At5g67460 Glycosyl hydrolase family protein 17Peri control Peri N-treat Peri N-treat AGI ID Peri control Peri N-treatMSX MSX MSX Gln At1g03170 6.629333333 8.987 6.908 7.8983333337.536333333 At1g03780 6.445 8.251666667 6.242666667 7.076 7.3 At1g079707.720333333 9.912 7.443 7.775 9.038666667 At1g10640 6.367 8.7993333336.453 7.744333333 6.997 At1g11730 6.103666667 8.170333333 6.3603333336.594333333 7.754 At1g03170 6.629333333 8.987 6.908 7.8983333337.536333333 At1g03780 6.445 8.251666667 6.242666667 7.076 7.3 At1g125706.595333333 8.687666667 6.505666667 7.758333333 7.168666667 At1g143506.290333333 7.305 6.303 6.201333333 6.440333333 At1g15570 6.1236666677.609666667 6.220333333 6.519333333 6.968 At1g17110 7.719 9.454 7.3488.486333333 7.590333333 At1g22180 7.009333333 8.553333333 6.9256666677.933666667 7.593666667 At1g24260 5.292333333 6.992666667 5.5576.047333333 6.253333333 At1g25510 6.964666667 8.999666667 6.6206666677.065333333 7.528 At1g26330 5.851666667 7.893333333 6.1553333336.869666667 6.676333333 At1g27370 6.157333333 8.466333333 6.7333333337.161333333 7.027 At1g30490 6.891666667 9.640666667 6.964 7.6383333337.654 At1g32930 7.046333333 7.695 6.801 6.958333333 7.184333333At1g35780 8.381333333 9.866666667 8.255 8.413 9.313666667 At1g48100 6.779.591 6.726333333 7.657333333 8.045333333 At1g49430 6.0396666678.640666667 6.123333333 6.694333333 6.830333333 At1g51790 7.1669.300333333 7.705333333 8.463666667 8.026666667 At1g52200 6.1366666677.614 6.267 7.199 6.628333333 At1g55690 6.831 8.371333333 6.7657.266666667 7.062 At1g62360 6.023333333 8.045333333 6.272666667 6.8067.001 At1g63470 8.112333333 9.699 8.085666667 8.629666667 7.918666667At1g65370 6.591666667 8.197666667 6.938333333 7.355 7.414 At1g673206.701666667 8.168666667 6.232333333 6.792333333 7.177666667 At1g707107.179333333 9.242666667 7.381 7.489666667 8.201666667 At1g72250 5.7678.719666667 5.833666667 6.353 6.592666667 At1g73930 7.0876666678.759666667 7.102666667 7.332 7.204333333 At1g74420 5.8106666677.516666667 5.918333333 6.254333333 6.341333333 At1g75240 8.08533333311.25466667 7.581666667 8.741666667 9.291666667 At1g76420 5.4553333337.892 5.602 6.613 6.456666667 At1g77110 6.595666667 7.818333333 6.5877.221333333 7.107 At1g77720 5.879333333 6.997666667 5.942666667 5.9946.402333333 At1g79350 7.392333333 9.181333333 7.391 7.5873333337.927666667 At1g79420 7.399 9.524 6.980666667 8.409666667 7.789At2g01210 5.557 7.320666667 5.092666667 5.822333333 5.971 At2g025406.500666667 9.078666667 6.758 7.721666667 7.203 At2g07170 6.5637.631666667 6.427 6.910333333 6.443 At2g07690 6.706 8.9 6.5243333337.609333333 7.002333333 At2g16250 5.965 7.485 6.353 6.501 6.680333333At2g17930 7.582 8.572 7.718333333 8.139666667 7.686333333 At2g201006.199666667 5.931666667 5.639333333 5.839333333 6.242666667 At2g203007.021666667 8.982 6.697333333 6.744333333 7.749 At2g21050 7.1043333339.603666667 6.763 7.399333333 7.186666667 At2g23700 7.433 9.5066666677.418 8.189333333 8.070666667 At2g25060 6.901666667 8.078 6.8813333337.278666667 7.28 At2g26180 7.388666667 9.992333333 7.1473333338.180666667 8.475333333 At2g26330 5.878 7.179666667 6.211666667 6.686.429666667 At2g27040 7.801 9.422 7.734333333 7.647666667 7.523333333At2g27980 6.429666667 8.005333333 6.546666667 6.980333333 6.760333333At2g31320 6.761 8.918 6.558 6.625666667 7.365 At2g32590 6.3086666677.441 6.624 6.633666667 7.205666667 At2g33560 6.314666667 7.8933333336.188333333 6.793666667 6.855 At2g34710 7.198666667 9.3943333337.274333333 7.806333333 7.735666667 At2g35340 7.081333333 9.5897.323666667 8.883 7.776666667 At2g36200 7.673666667 9.2856666677.203333333 8.401 7.974666667 At2g38160 5.758666667 8.3033333335.999333333 6.653 6.507666667 At2g42120 6.645666667 8.156333333 6.2446.578333333 7.092666667 At2g44440 6.682 8.576333333 6.836 7.3763333337.466333333 At2g44830 7.164666667 9.854 7.141666667 8.0496666677.700666667 At2g45870 5.999333333 6.984 6.218 6.518 6.576 At3g021106.842333333 8.848666667 6.744333333 7.120666667 7.499 At3g02210 9.48812.07133333 9.066666667 9.991666667 10.617 At3g02640 7.1263333338.286666667 6.631666667 7.316333333 7.360666667 At3g05750 5.134 5.9495.267666667 5.435 5.833333333 At3g06130 7.779 8.985333333 8.1877.799666667 7.110666667 At3g06220 5.238333333 6.497333333 5.2806666675.806 5.888 At3g10310 5.342666667 6.054 5.204333333 5.406 5.748666667At3g11000 5.394333333 7.509333333 5.627 5.956666667 6.068 At3g130007.239 9.596333333 7.339666667 8.044 7.802 At3g13510 6.904 8.8126.918666667 7.554 7.346333333 At3g13510 6.904 8.812 6.918666667 7.5547.346333333 At3g14980 6.367666667 8.789 6.775333333 6.863333333 7.021At3g15550 6.392333333 8.608333333 6.245666667 6.869333333 7.248At3g16170 6.768666667 7.89 6.775 7.220333333 7.347 At3g17840 6.6826666679.281333333 6.523666667 6.638666667 7.281333333 At3g20070 6.3137.989333333 6.540333333 6.800666667 6.797333333 At3g21310 5.8886666678.052333333 5.685333333 6.425333333 6.586 At3g26932 4.8413333336.968333333 4.767333333 5.455666667 5.767333333 At3g29280 6.5167.565666667 6.103666667 7.068 6.996 At3g32400 5.581 6.4243333336.088333333 6.019666667 6.224666667 At3g45610 5.646 7.9423333335.479666667 6.05 6.331 At3g50890 6.034666667 8.649 5.9036666676.850666667 6.776666667 At3g57670 6.422 9.234666667 6.275 7.4363333337.342666667 At3g57830 6.463333333 8.503333333 6.479 6.9326666677.188666667 At3g57920 6.385666667 7.443666667 6.186 6.3336666676.672666667 At3g61310 6.532333333 8.044666667 6.731333333 7.1363333337.072666667 At4g02800 6.794333333 8.853666667 6.288666667 7.5646666677.206333333 At4g11450 6.694333333 8.830333333 6.832666667 7.217.621666667 At4g13710 6.084666667 8.050666667 5.720333333 5.9676.635666667 At4g14330 5.899333333 7.957 5.962 6.768333333 6.675At4g17000 6.014333333 7.355666667 6.114333333 5.939 6.901333333At4g18020 7.304666667 9.041666667 7.239666667 7.917 7.697 At4g188206.655666667 7.926333333 6.827333333 7.292666667 6.945666667 At4g213265.717 6.696 6.009 6.650333333 6.482 At4g21430 6.623666667 8.9113333336.861 6.803 7.628666667 At4g21550 6.343666667 8.313666667 6.6213333337.009666667 7.127666667 At4g25110 6.056666667 8.337666667 6.2063333337.327 6.588666667 At4g29030 6.849666667 8.580333333 6.6376666677.178333333 7.343 At4g30130 5.364666667 6.309333333 5.577 5.7703333336.023666667 At4g32730 9.842333333 11.35666667 9.267333333 9.76766666710.273 At4g37750 6.221 7.476333333 6.530333333 6.756333333 6.923666667At4g39010 6.672666667 8.810333333 6.708666667 7.605333333 7.294666667At5g02370 5.355333333 6.996333333 5.774333333 6.533333333 6.114333333At5g03680 5.81 7.621 5.925 6.446333333 7.164333333 At5g07180 6.5219.025333333 6.564333333 7.577333333 7.381333333 At5g07800 5.7266666676.798333333 5.929 6.02 6.394666667 At5g08390 8.283 10.445666678.063333333 9.216666667 9.263 At5g11160 6.866 7.75 6.56 6.6316666677.227 At5g11510 6.458333333 8.032 6.629666667 6.909666667 7.412333333At5g20540 9.235333333 11.241 8.630333333 9.738333333 9.211333333At5g20740 7.079 10.15533333 6.266 7.398333333 8.301666667 At5g250906.363666667 8.895666667 6.323 7.242666667 7.277666667 At5g268507.424666667 9.447333333 7.543666667 8.227666667 7.769 At5g276804.872333333 7.236666667 5.111333333 5.733666667 6.488333333 At5g333705.896666667 6.865666667 6.042 6.207333333 6.621333333 At5g359307.115333333 8.889666667 7.311 8.176 7.686 At5g37020 6.606333333 8.026.775333333 7.254666667 7.422 At5g43080 6.434666667 8.283666667 6.3397.162666667 7.131 At5g51560 6.200333333 8.106666667 5.6686666675.713666667 6.312333333 At5g52860 6.35 8.370666667 6.596 6.8183333337.600333333 At5g56740 8.164 10.43366667 7.634333333 8.967666667 8.365At5g60210 7.806333333 9.165333333 8.343333333 8.125333333 7.937666667At5g60910 6.354333333 8.646333333 6.7 7.529666667 7.323333333 At5g649804.968666667 6.349 5.462 5.691666667 5.688 At5g67110 5.832666667 7.2885.959333333 6.354 6.560666667 At5g67460 6.212 7.625666667 6.337 6.7246.992

To test whether the putative ARF8 module formed a cohesive responsegroup, we asked whether ARF8 and the 126 potential targets respondedsimilarly to either nitrate or downstream metabolites. Thus, we treatedroots with nitrate and methionine sulfoximine (MSX), which blocks theassimilation of nitrate into glutamine and consequently glutamate (Rawatet al., 1999, Plant J 19:143-152), and collected pericycle cells for RNAanalysis. Induction of ARF8 and all 126 of the putative ARF8 targets wasblocked by MSX treatment (q<0.05 FDR), suggesting they were responsiveto downstream nitrogen metabolites rather than nitrate itself (FIG. 3M).To confirm that the effect was specific to metabolite signaling, werepeated the MSX block of nitrate metabolism into glutamate/glutamine,but added glutamine, which should restore metabolite signaling if thesignal is glutamine or a derived nitrogen metabolite. The induction ofARF8 and all 126 of the putative ARF8 targets was indeed restored by theglutamine “add back” (q<0.05 FDR) (FIG. 3M). No other ARFs that wereinduced in the pericycle showed the same coordinated regulation withthis cluster. Overall, the data is consistent with ARF8 and its putativepericycle targets forming a cohesive response module under coordinatedregulation by glutamine or a downstream metabolite (FIG. 3N).

Together this work suggests that the Arabidopsis root undergoes aconcerted and rapid response to nitrate which is highly cell-specific.Arabidopsis metabolism appears to be coordinated in all cells of theroot while simultaneously developmental processes such as lateral rootdevelopment are regulated at the cell-specific level by N-assimilationproducts. We have revealed a mechanism by which the root regulates itsbranching according to levels of nitrogen which likely intersects withauxin-regulation of this process. Our results suggest a novel pathwayfor regulating the balance between lateral root initiation andemergence, a distinction which has not so far been examined in mutants(Malamy et al., 2005, Plant Cell Environ 28, 67-77). We have alsorevealed that microRNAs could act to mediate development according tonutrients, implying a new layer of developmental regulation bynutrients.

8. Equivalents

Although the invention is described in detail with reference to specificembodiments thereof, it will be understood that variations which arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference in their entireties.

What is claimed is:
 1. A method of producing a transgenic plant havingan improved agronomic or nutritional characteristic, said methodcomprising overexpressing miR167 specifically in the pericycle of theplant by transforming the plant with a polynucleotide constructcomprising a sequence encoding miR167 operably linked to apericycle-specific promoter, wherein the promoter is optionally linkedto an inducible promoter element, wherein the one or more agronomic ornutritional characteristics is a(n): (a) increased lateral rootformation, (b) increased surface area of roots, (c) increased root mass,and/or (d) altered nitrogen response wherein the agronomic ornutritional characteristic is improved in the transgenic plant ascompared to a wild type plant.
 2. A method of producing a transgenicplant, comprising: transforming a plant with a polynucleotide constructcomprising a sequence encoding miR167 operably linked to apericycle-specific promoter, wherein the promoter is optionally linkedto an inducible promoter element; and identifying a transgenic plantoverexpressing miR167 in the pericycle from among transgenic plantshaving the polynucleotide construct.
 3. The method of claim 1, whereinthe transgenic plant and wild type plant are cultivated under conditionsin which apical root growth is repressed in the wild type plant.
 4. Themethod of claim 1, wherein the transgenic plant and wild type plant arecultivated under nitrogen-moderate or nitrogen-rich conditions.
 5. Amethod of producing a transgenic plant having decreased ARF8-mediatednitrogen-responsiveness, said method comprising overexpressing miR167specifically in the pericycle of the plant by transforming the plantwith a polynucleotide construct comprising a sequence encoding miR167operably linked to a pericycle-specific promoter, wherein the promoteris optionally linked to an inducible promoter element, wherein theARF8-mediated nitrogen responsiveness is decreased in the transgenicplant as compared to a wild type plant.
 6. A method of producing atransgenic plant having decreased ARF8-mediated nitrogen responsiveness,comprising: transforming a plant with a polynucleotide constructcomprising a sequence encoding miR167 operably linked to apericycle-specific promoter, wherein the promoter is optionally linkedto an inducible promoter element; identifying a transgenic plantoverexpressing miR167 in the pericycle from among transgenic plantshaving the polynucleotide construct, screening the transgenic plantoverexpressing miR167 for decreased ARF-8-mediated nitrogenresponsiveness as compared to a wild type plant; and selecting thetransgenic plant having decreased ARF8-mediated nitrogen-responsiveness.7. The method of claim 5 or 6, wherein the transgenic plant and wildtype plant are cultivated under conditions in which apical root growthis repressed in the wild type plant.
 8. The method of claim 5 or 6,wherein the transgenic plant and wild type plant are cultivated undernitrogen-moderate or nitrogen-rich conditions.
 9. The method of claim 1,2, 5 or 6, wherein the plant is species of woody, ornamental,decorative, crop, cereal, fruit, or vegetable.
 10. The method of claim1, 2, 5 or 6, wherein said plant is a species of one of the followinggenuses: Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium,Arabidopsis, Arachis, Beta, Betula, Brassica, Capsicum, Ceratopteris,Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus,Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca,Linum, Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago,Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus,Physcomitrella, Picea, Pinus, Poncirus, Populus, Prunus, Robinia, Rosa,Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia,Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia.11. A transgenic plant produced by a method as claimed in claim
 2. 12. Atransgenic plant comprising a polynucleotide construct comprising asequence encoding miR167 operatively linked to a pericycle-specificpromoter, wherein the promoter is optionally linked to an induciblepromoter element.
 13. The transgenic plant of claim 12, wherein theplant is species of woody, ornamental, decorative, crop, cereal, fruit,or vegetable.
 14. The transgenic plant of claim 12, wherein said plantis a species of one of the following genuses: Acorus, Aegilops, Allium,Amborella, Antirrhinum, Apium, Arabidopsis, Arachis, Beta, Betula,Brassica, Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas,Descurainia, Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis,Helianthus, Hordeum, Ipomoea, Lactuca, Linum, Liriodendron, Lotus,Lupinus, Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar,Pennisetum, Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus,Populus, Prunus, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum,Solanum, Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria,Triticum, Vitis, Zea, or Zinnia.
 15. A transgenic plant-derivedcommercial product, which product is derived from the transgenic plantof claim 11, 12, 13 or 14, and wherein said product contains thepolynucleotide construct.
 16. The transgenic plant-derived commercialproduct of claim 15, wherein said transgenic plant is a tree, and saidcommercial product is pulp, paper, a paper product, or lumber.
 17. Thetransgenic plant-derived commercial product of claim 15, wherein saidtransgenic plant is tobacco, and said commercial product is a cigarette,cigar, or chewing tobacco.
 18. The transgenic plant-derived commercialproduct of claim 15, wherein said transgenic plant is a crop, and saidcommercial product is a fruit or vegetable.
 19. The transgenicplant-derived commercial product of claim 15, wherein said transgenicplant is a grain, and said commercial product is bread, flour, cereal,oat meal, or rice.
 20. An expression vector comprising a polynucleotideencoding miR167 operatively linked to a pericycle-specific promoter. 21.The method of claim 2 further comprising screening the transgenic plantoverexpressing miR167 for an improved agronomic or nutritionalcharacteristic under nitrogen-rich growth conditions as compared to awild type plant, wherein the agronomic or nutritional characteristic isselected from the group consisting of increased lateral root formation,increased surface area of roots, increased root mass, and alterednitrogen response; and selecting the transgenic plant having theimproved agronomic or nutritional characteristic.
 22. A transgenic plantproduced by a method as claimed in claim 1 or
 5. 23. A seed derived fromthe transgenic plant of claim 11 or 12, wherein said seed comprises saidconstruct.
 24. A transgenic plant-derived commercial product, whichproduct is derived from the transgenic plant of claim 22, and whereinthe product contains the transgenic modification.
 25. A transgenic plantproduced by a method as claimed in claim
 6. 26. A transgenicplant-derived commercial product, which product is derived from thetransgenic plant of claim 25, and wherein said product contains thepolynucleotide construct.
 27. A seed derived from the transgenic plantof claim 25, wherein said seed comprises said construct.